Systems and methods for optically processing samples

ABSTRACT

A system for processing a sample includes a chamber having at least one inlet and at least one outlet, where the chamber is configured to accommodate flow of the sample from the at least one inlet toward the at least one outlet, and an imager array configured to image the flow of the sample in the chamber, where the imager array includes at least one lensless image sensor configurable opposite at least one light source.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 62/859,666 filed Jun. 10, 2019, U.S. Provisional Application Ser.No. 62/800,385 filed Feb. 1, 2019, and U.S. Provisional Application Ser.No. 62/742,833 filed on Oct. 8, 2018, each of which is herebyincorporated by reference in its entirety.

TECHNICAL FIELD

This invention relates generally to the field of assays for processingsample entities.

BACKGROUND

Devices to conduct assays are commonly used for the purposes ofbiochemistry research, pharmaceutical discovery, cell screening, medicaldiagnostics, and other applications to detect and/or measure one or morecomponents of a sample. A digital assay is one kind of assay thatpartitions a biological sample into multiple smaller containers suchthat each container contains a discrete number of biological entities.For example, a digital assay may be used to analyze microfluidicdroplets including single cells or other entities, such as forquantifying nucleic acids, proteins, or other biological content.

Current microfluidic systems have a number of drawbacks. For example,conventional microfluidic digital assays require that droplets bemonodisperse and of the same type (e.g., exclusively DNA) during anexperiment, in order to, for example, accurately correlate measurementsto analyte concentration and compare such measurements across differentdroplets. These devices require droplets to be pre-sorted to ensure thatthey are of suitably uniform size, which is time-consuming and reducesefficiency in processing droplets. Additionally, these devices include alinear, single-track microfluidic channel within which droplets travelin series for processing, which further limits the efficiency foranalysis of the droplets. Accordingly, there is a need for new andimproved digital assay systems and methods for processing samples.

SUMMARY

Generally, a system for processing a sample may include a chamber havingat least one inlet and at least one outlet, where the chamber isconfigured to accommodate flow of the sample from the at least one inlettoward the at least one outlet. The system may further include an imagerarray configured to image the flow of the sample in the chamber, wherethe imager array includes at least one lensless image sensorconfigurable opposite at least one light source. In some variations, thechamber may be configured to accommodate a two-dimensional flow of thesample, such as movement in multiple directions (e.g., within an X-Yplane of the chamber). The imager may include a two-dimensional array oflensless image sensors for imaging sample flow in the chamber. Asanother example, the imager may include a one-dimensional or single-linearray of lensless image sensors for imaging sample flow in the chamber.By being located opposite at least one light source across the chamber,the imager array may, in some variations, be configured to generateshadow images of the flow of the sample in the chamber.

The chamber may include opposing surfaces that are offset to form aspacing that receives sample flow. For example, the chamber may includea first surface and a second surface that is offset from the firstsurface. A plurality of spacers may be disposed between the first andsecond surfaces (e.g., to enforce and/or support the spacing between thefirst and second surfaces). At least one of the first surface and secondsurface may include an optically transparent material (e.g., polyimide,glass, etc.). At least one of the first surface and the second surfacemay be formed through planar processing techniques such as semiconductormanufacturing processes. The first surface and the second surface may beconfigured to flatten at least a portion of the sample, such thatflattened samples or sample entities, as will be described in furtherdetail herein, may flow through the chamber.

In some variations, the system may further include a light source, wherethe imager array and the light source are opposing each other across thechamber. The imager array may be embedded in a first structure having afirst optically transparent portion adjacent the chamber. The lightsource may be embedded in a second structure having a second opticallytransparent portion adjacent the chamber.

Generally, another variation of a system for processing a sample mayinclude a chamber defined at least partially by a first structure and asecond structure opposing the first structure, where each of the firstand second structures has at least a portion that is opticallytransparent. The system may further include at least one light sourcethat is embedded in the first structure and configured to emit lighttoward the chamber, and an imager array embedded in the second structureand configured to image the chamber. The imager array may include atleast one lensless image sensor. The imager array may include aone-dimensional or two-dimensional array of lensless image sensors. Theimager array may be configured to generate shadow images of the flow ofthe sample. In some variations, the first structure and the secondstructure may be integrally formed.

The chamber may be configured to accommodate a two-dimensional flow ofthe sample between at least one inlet and at least one outlet of thechamber. The chamber may be configured to flatten at least a portion ofthe sample (e.g., between the opposing first and second structures). Insome variations, a plurality of spacers may be disposed in the chamberbetween the first structure and second structure. At least one of suchspacers may include an anchor bonding the first structure and the secondstructure together. For example, in some variations the anchor mayinclude solder, polymer adhesive, or other suitable anchor material thatmay flow into one or more vias in a spacer and adjoin facing surfaces ofthe first and second structures.

In some variations, at least one of the first structure and secondstructure may include a laminated stack up of optically transparentlayers. For example, at least one of the first structure and the secondstructure may be formed through planar processing.

The sample may, in some variations, include at least one POD as furtherdescribed herein. At least one POD may include an analyte, such as acell, DNA, RNA, a nucleotide, a protein, and/or an enzyme. Additionallyor alternatively, at least one POD may lack, or not include, an analyte.In use, the assay system may be used to generate optical images of PODSand their contents, to generate information from which chemical and/orbiological information may be derived.

Generally, in some variations, a system for processing a sampleincluding a plurality of particles may include a chamber configured toaccommodate the sample, where the chamber includes at least oneelectrode configured to deliver electrical energy sufficient to merge aselected portion of particles in the sample, and a sorting arrangementconfigured to separate particles of the sample based on particle size.For example, in some variations the chamber may include a plurality ofelectrodes extending between first and second opposing surfaces of thechamber (e.g., may provide structural support in combination withelectrode functionality). The chamber may be configured to accommodate atwo-dimensional flow of the sample. Furthermore, in some variations thesystem may further include an imager array (e.g., including a lenslessimage sensor) configured to generate one or more images of the sample inthe chamber, and a controller configured to activate the at least oneelectrode to deliver electrical energy to the selected portion ofparticles based on the one or more images of the sample.

In some variations, the sorting arrangement may include a passivesorting arrangement. For example, the sorting arrangement may include aplurality of spacers. The spacers may be arranged in a staggered arrayand configured to perform particle separation via deterministic lateraldisplacement. As another example, the chamber may include a first outletand a second outlet, where the first outlet is sized to passsubstantially only particles below a predetermined threshold particlesize, and the second outlet may be sized to pass particles above thepredetermined threshold particle size. Additionally or alternatively,the chamber may include a plurality of branching channels configured toperform particle separation via hydrodynamic filtration. Additionally oralternatively, in some variations the chamber may include an activesorting arrangement (e.g., via active fluidic control, PDEP forces,etc.).

Generally, in some variations a system for processing a sample mayinclude a chamber configured to accommodate the flow of a sample wherethe chamber includes at least one electrode configured to selectivelydeliver electrical energy to at least a portion of the sample, an imagerarray (e.g., including a lensless image sensor) configured to image theflow of the sample in the chamber, and a controller configured toactivate the at least one electrode based on an analysis of the one ormore images.

In some variations, the chamber may be configured to accommodate atwo-dimensional flow of the sample. The chamber may include a pluralityof electrodes, and the controller may be configured to selectivelyactivate pairs of electrodes, such as adjacent electrodes. The activatedelectrodes may, for example, be capacitively coupled with one or moretarget particles, which may cause the target particles to merge.

In some variations, the system may further include a sorting arrangementconfigured to separate particles of the sample based on particle size.The sorting arrangement may include a passive sorting arrangement. Thesorting arrangement may, for example, include a plurality of spacersarranged in a staggered array and configured to perform particleseparation via deterministic lateral displacement. As another example,the chamber may include a first outlet and a second outlet, where thefirst outlet is sized to pass only particles below a predeterminedthreshold particle size, and the second outlet may be sized to passparticles above the predetermined threshold particle size. Additionallyor alternatively, the chamber may include a plurality of branchingchannels configured to perform particle separation via hydrodynamicfiltration. Additionally or alternatively, in some variations thechamber may include an active sorting arrangement (e.g., via activefluidic control, PDEP forces, etc.).

Generally, in some variations, a method for processing a sampleincluding a plurality of particles (e.g., PODS) may include receiving asample in a chamber including at least one electrode, characterizing oneor more particles in the sample as discard particles, merging thediscard particles by delivering electrical energy from the at least oneelectrode to the discard particles, and sorting particles of the samplebased on particle size. In some variations, characterizing one or moreparticles may include receiving one or more images of the sample in thechamber and characterizing one or more particles based on the one ormore images. The one or more images may include, for example, an opticalshadow image of the sample.

In some variations, delivering electrical energy may include activatinga pair of electrodes in accordance with a drive waveform. The drivewaveform may, for example, be an AC waveform. The waveform may, in somevariations, have a peak-to-peak voltage of between about 0.5 V and about10 V, or between about 0.5 V and about 5 V. Furthermore, in somevariations, the waveform may have a frequency between about 1 Hz and 1MHz, or between about 50 Hz and about 20 kHz.

In some variations, sorting particles may include passively sorting theparticles. For example, particles may be sorted via deterministiclateral displacement. As another example, particles may be sorted bypermitting particles of a first size to pass through a first outlet ofthe chamber, and permitting particles of a second size to pass through asecond outlet of the chamber. Additionally or alternatively, particlesmay be sorted via hydrodynamic filtration.

Furthermore, the method may in some variations be used to process asample in which at least a portion of the particles contains one or morecells (e.g., CHO cells, hybridomas, B cells, myeloma cells, etc.)secreting a substance of interest (e.g., antibody, insulin, etc.). Inthese variations, characterizing one or more particles in the sample mayinclude characterizing secretion levels of the one or more cells, suchas by characterizing agglutination in the one or more cells. Forexample, particles lacking secretor cells and/or particles containinglow secretor cells may be characterized as discard particles, whileparticles including high secretor cells may be characterized asparticles of interest. Particles for discard and particles of interestmay be sorted and separated. For example, sorting may include sortingparticles below a threshold size as particles of interest (e.g.,particles containing high secretor cells). In some variations, thesample may be prepared such that there is an average of about 0.1 cellsper particle.

Generally, in some variations, a system for enabling selection of a cellof interest from a population of cells may include an encapsulationreagent, where the encapsulation reagent has a density greater thanabout 1.0, and a first plurality of particles suspended in aqueousmedia, where each particle of the first plurality of particles includesa first binding partner that is specific to a second binding partnersecreted by the cell of interest. In some variations, the encapsulationreagent may include a surfactant. In some variations, the surfactantincludes at least one of fluorine and polyethylene glycol. In somevariations, each particle of the first plurality of particles may have adiameter between about 30 nm to about 50 In some variations, eachparticle of the first plurality of particles may include at least one ofpolystyrene, gold, cellulose, latex, agarose, polyethylene glycol (PEG),glass, and magnetic beads. In some variations, a first cluster site isformed by a binding of the first and second binding partners. In somevariations, the first binding partner and the second binding partner maybe a first and second protein. In these variations, the first bindingpartner or the second binding partner may be an antigen or antibody. Forexample, the antibody may be IgG. In some variations, the first bindingpartner and the second binding partner may be a first and secondpeptide.

Furthermore, the system in some variations may also include a secondplurality of particles, where each particle of the second plurality ofparticles has a third binding partner that is specific to a fourthbinding partner secreted by the cell of interest. In these variations,the system may further include a second cluster site formed by a bindingof the third and fourth binding partners.

Generally, in some variations, a mixture may include an encapsulationreagent, one or more first particles suspended in aqueous media, whereeach first particle includes a first binding partner, and a populationof cells with at least one cell of interest that secretes a protein ofinterest having a second binding partner, where the first bindingpartner is specific to the second binding partner. In some variations,the encapsulation reagent may include a surfactant. In some variations,the surfactant includes at least one of fluorine and polyethyleneglycol. In some variations, the encapsulation reagent may be betweenabout 60% and 90% of the mixture by volume. In some variations, the oneor more first particles may be between about 5% and 20% of the mixtureby volume. In some variations, the population of cells may be betweenabout 5% and 20% of the mixture by volume. In some variations, eachparticle of the first plurality of particles may have a diameter betweenabout 30 nm to about 50 In some variations, each particle of the firstplurality of particles may include at least one of polystyrene, gold,cellulose, latex, agarose, polyethylene glycol (PEG), glass, andmagnetic beads. In some variations, the mixture may further include afirst cluster site formed by a binding of the first and second bindingpartners. In some variations, the first binding partner and the secondbinding partner may be a first and second protein. In these variations,the first binding partner or the second binding partner may be anantigen or antibody. For example, the antibody may be IgG. In somevariations, the first binding partner and the second binding partner maybe a first and second peptide. In some variations, the population ofcells may include at least one or more of CHO cells, B cells, hybridomacells, plasma cells, HEK293 cells, myeloma cells, and T cells. In somevariations, the one or more first particles may include one or morecells, and the first binding partner may include antigens expressed onthe one or more cells. In some variations, the first plurality ofparticles may include a second population of cells, and the firstbinding partner may include antigens expressed on the second populationof cells.

Furthermore, in some variations, the mixture may also include aplurality of sample entities, where each sample entity encapsulates atleast one or more of the one or more first particles, at least one cellfrom the population of cells, and the aqueous media. In thesevariations, the plurality of sample entities may be polydisperse sampleentities.

Furthermore, the mixture in some variations may also include a secondplurality of particles, where each particle of the second plurality ofparticles has a third binding partner that is specific to a fourthbinding partner secreted by the at least one cell of interest. In thesevariations, the system may further include a second cluster site formedby a binding of the third and fourth binding partners.

Generally, in some variations, a method for preparing a sample for aclustering assay system may include providing a population of cellshaving at least one cell of interest, combining the population of cells,a first plurality of particles, and an encapsulation reagent to create amixture, where each particle of the first plurality of particles issuspended in aqueous media and includes a first binding partner that isspecific to a second binding partner secreted by the at least one cellof interest; and agitating the mixture to create an emulsion, therebyencapsulating the population of cells into a plurality of polydispersesample entities (e.g., PODS). In some variations, the first bindingpartner and the second binding partner may be a first and secondprotein. In these variations, the first binding partner or the secondbinding partner may be an antigen or antibody. For example, the antibodymay be IgG. In some variations, the first binding partner and the secondbinding partner may be a first and second peptide. In some variations,the population of cells may include at least one or more of CHO cells, Bcells, hybridoma cells, plasma cells, HEK293 cells, myeloma cells, and Tcells. In some variations, the first plurality of particles may includea second population of cells, and the first binding partner may includeantigens expressed on the second population of cells.

In some variations, providing the population of cells may includediluting the population of cells to obtain a desired cell concentrationof between about 100,000 and 300,000 cells per milliliter. In thesevariations, the desired cell concentration may be about 220,000 cellsper milliliter.

Furthermore, in some variations, combining the population of cells, thefirst plurality of particles and the encapsulation reagent may alsoinclude adding a second plurality of particles to form the mixture,where each particle of the second plurality of particles comprises athird binding partner that is specific to a fourth binding partnersecreted by the at least one cell of interest. In these variations, thesecond binding partner and the fourth binding partner may be a firstcomponent and a second component of an antibody, respectively.

Furthermore, in some variations, the emulsion may be characterized by aλ value, where λ is a number of cells per sample entity of the pluralityof polydisperse sample entities. In these variations, the λ value may bebetween about 0 and about 10 cells per sample entity.

Furthermore, the method in some variations may also include incubatingthe emulsion for a predetermined length of time. In these variations,the predetermined length of time may be between about 1 and about 6hours.

Generally, in some variations, a method for selecting at least one cellof interest from a population of cells may include providing an emulsionhaving the population of cells and a first plurality of particles, wherethe population of cells and the first plurality of particles areencapsulated into a plurality of polydisperse sample entities (e.g.,PODS), and where each particle of the first plurality of particles issuspended in aqueous media and includes a first binding partner that isspecific to a second binding partner secreted by the at least one cellof interest, measuring a signal for at least one sample entity, wherethe signal is at least partially associated with binding of the firstand second binding partners; and identifying the at least one cell ofinterest based at least in part on the measured signal. In somevariations, the second binding partner may be coupled to a firstcomponent of a protein of interest secreted by the at least one cell ofinterest, and where the measured signal quantifies the protein ofinterest in the at least one sample entity. In some variations, thefirst plurality of particles may include a second population of cells,and the first binding partner may include antigens expressed on thesecond population of cells.

In some variations, the emulsion may also include a second plurality ofparticles encapsulated into the plurality of polydisperse sampleentities (e.g., PODS), where each particle of the second plurality ofparticles includes a third binding partner that is specific to a fourthbinding partner secreted by the at least one cell of interest. In thesevariations, the signal may at least partially be associated with abinding of the first and second binding partners, and may at leastpartially be associated with a binding of the third and fourth bindingpartners. In these variations, the second binding partner and the fourthbinding partner may be associated with a protein of interest secreted bythe at least one cell of interest, and the measured signal may quantifybinding affinity and/or specificity of the protein of interest to thefirst binding partner or the third binding partner. In these variations,the measured signal may quantify antigen binding affinity and/orspecificity of an antibody secreted from a cell of interest.

In some variations, identifying the at least one cell of interest mayinclude identifying at least a portion of the sample entities that has ameasured signal greater than a predetermined threshold. In somevariations, measuring the signal for the at least one sample entity mayinclude receiving at least one shadow image of the at least one sampleentity, and determining a size score of at least one object in thesample entity based on the at least one shadow image, where the measuredsignal is based at least in part on the size score.

Furthermore, in some variations, the method may also include introducingthe emulsion into a chamber adjacent an imager array configured togenerate the at least one shadow image.

Furthermore, in some variations, the method may also include removing atleast one cell of interest from the polydisperse sample entities. Inthese variations, the method may also include analyzing the at least onecell of interest with one or more of PCR, FACS, DNA sequencing, andELISA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict schematic illustrations of exemplary variationsof an assay system for optically processing samples;

FIG. 2A depicts a schematic illustration of a chamber arrangement with alensless image sensor;

FIG. 2B depicts an exemplary shadow image obtained with a lensless imagesensor in the chamber arrangement of FIG. 2A.

FIG. 3 depicts an exemplary variation of an assay system for opticallyprocessing samples.

FIGS. 4A-4D depict schematic illustrations of an exemplary variation ofa chamber arrangement. FIG. 4A depicts a cross-sectional view of avariation of a chamber arrangement. FIG. 4B depicts an exploded view ofa portion of the chamber arrangement depicted in FIG. 4A. FIG. 4Cdepicts a cross-sectional view of the portion of the chamber arrangementdepicted in FIG. 4B. FIG. 4D is a partial top plan view of the chamberarrangement depicted in FIG. 4A.

FIG. 5 depicts a detailed partial cross-sectional view of the chamberarrangement depicted in FIG. 4A.

FIG. 6 depicts another detailed partial cross-section view of thechamber arrangement depicted in FIG. 4A.

FIGS. 7A and 7B depict schematic illustrations of another exemplaryvariation of a chamber arrangement. FIG. 7A depicts a cross-sectionalview of a variation of a chamber arrangement. FIG. 7B depicts a detailedpartial cross-sectional view of the chamber arrangement depicted in FIG.7A.

FIGS. 8A and 8B depict schematic illustrations of another exemplaryvariation of a chamber arrangement. FIG. 8A depicts a cross-sectionalview of a variation of a chamber arrangement. FIG. 8B depicts a top planview of the chamber arrangement depicted in FIG. 8A.

FIG. 9 depicts an exemplary image taken with a lensless image sensor inan exemplary variation of an assay system for optically processingsamples.

FIG. 10 depicts another exemplary image taken with a lensless imagesensor in an exemplary variation of an assay system for opticallyprocessing samples.

FIGS. 11A and 11B depict schematic illustrations of another exemplaryvariation of a chamber arrangement with a lensless image sensor.

FIG. 12 depicts a chart showing exemplary assay types that may beperformed by the assay system.

FIG. 13A provides an exemplary image of computer vision techniques todetect PODS and beads in PODS. FIG. 13B is an illustrative graph ofdistributions of detected particle size scores from samples includingvarious-sized beads.

FIGS. 14A-14C provide exemplary images of computer vision techniques fordetecting PODS and protein agglutinates in samples containing threedifferent protein concentrations. FIG. 14A is an exemplary image ofcomputer vision detection of PODS containing IgG at a concentration of 0ng/mL. FIG. 14B is an exemplary image of computer vision detection ofPODS containing IgG at a concentration of 30 ng/mL. FIG. 14C is anexemplary image of computer vision detection of PODS containing IgG at aconcentration of 480 ng/mL.

FIGS. 15A-15C are illustrative graphs of the distribution of multipleparameters of PODS at various protein concentrations detected usingcomputer vision techniques. FIG. 15D is an illustrative bar graphshowing the number of POD detected at each protein concentration. FIGS.15E-15H are illustrative graphs of the distribution of BE scores, whichare calculated using one or more parameters of agglutinates within thePODS and/or POD characteristics, at various protein concentrations.

FIGS. 16A-16D are illustrative graphs of mean and median of various BEscores that have been correlated with protein concentration.

FIGS. 17A and 17B are illustrative graphs of BE scores related to theprecision of protein-based assays at various protein concentrations.

FIG. 18A provides an exemplary image of computer vision detection ofPODS containing a control sample. FIG. 18B provides an exemplary imageof computer vision detection of PODS containing bovine serum and asample of 960 ng/mL rabbit IgG. FIG. 18C is an illustrative graphcomparing the distribution of a POD parameter score in the control andthe 960 ng/mL samples.

FIG. 19A depicts an illustrative schematic of a CD-45+ cell tagged withanti-CD45 nanoparticles. FIG. 19B provides two exemplary images ofcomputer vision detection of PODS containing CD-45+ cells tagged asshown in FIG. 19A.

FIG. 20A provides an exemplary image of computer vision detection ofPODS containing yeast cells stained with trypan blue. FIG. 20B is anillustrative graph of the particle count scores of yeast cells detectedby computer vision. FIG. 20C is an illustrative graph of thedistribution of the particle size scores of the yeast cells detected bycomputer vision.

FIGS. 21A-21E illustrate an exemplary method for processing an image ofone or more PODS and identifying PODS in the processed image.

FIG. 22 depicts an illustrative schematic of another method forprocessing samples in a cell secretion assay.

FIG. 23A depicts a flowchart of an exemplary method for processingsamples with electromerging.

FIG. 23B depicts an illustrative schematic of an exemplary variation ofpreparing a sample.

FIG. 23C depicts an illustrative schematic of an exemplary variation ofa method for processing samples with electromerging.

FIGS. 23D and 23E illustrate exemplary variations of sortingarrangements in a chamber for processing samples.

FIG. 24A depicts an illustrative schematic of an exemplary variation ofan electromerging chamber arrangement. FIG. 24B depicts an illustrativeschematic of a cross-sectional stack-up of the variation of theelectromerging chamber arrangement depicted in FIG. 24A.

FIG. 24C depicts an illustrative schematic of an exemplary variation ofan electromerging chamber arrangement and a controller.

FIGS. 25A-25C are images illustrating exemplary hybridoma growth ratesover time, as viewed with imager systems described herein.

FIGS. 26A and 26B are images illustrating exemplary hybridoma secretionranges associated with low and high IgG concentrations, respectively, asviewed with imager systems described herein.

FIG. 27 depicts images illustrating detection of agglutination in anexemplary experiment to assess hybridoma secretion ranges after variousincubation periods.

FIGS. 28A and 28B depict another variation of electrodes in an exemplaryvariation of an electromerging chamber arrangement.

FIG. 29A is a table of system parameters for an exemplary electromergingchamber arrangement and exemplary sample with B cells. FIGS. 29B and 29Care an actual distribution and a modeled distribution, respectively, ofPOD sizes in the sample described in FIG. 29A.

FIG. 30 depicts an illustrative schematic of another exemplary variationof an electromerging chamber arrangement.

FIG. 31 depicts an illustrative schematic of an exemplary variation of asystem for processing samples with electromerging.

FIG. 32A depicts a schematic diagram of the binding interactions thatmay occur inside of a POD when using a one-bead assay. FIG. 32B shows adetailed enlargement of a region of FIG. 32A, showing a single particle.

FIG. 33A depicts a schematic diagram of the binding interactions thatmay occur inside of a POD when using a two-bead assay. FIG. 33B shows adetailed enlargement of a POD of FIG. 33A, and FIG. 33C shows a detailedenlargement of a binding interaction within the POD of FIGS. 33A-33B.

FIG. 34A depicts a flow chart showing an exemplary method of preparing asample for a one-bead clustering assay system. FIG. 34B depicts a flowchart showing an exemplary method of preparing a sample for a two-beadclustering assay system.

FIG. 35A depicts an exemplary method of selecting at least one cell ofinterest from a population of cells for use in a one-bead assay. FIG.35B depicts an exemplary method of selecting at least one cell ofinterest from a population of cells for use in a two-bead assay.

FIGS. 36A-36C depict 4× objective microscope cell images from tests of aone-bead assay. To demonstrate the one-bead assay, one batch ofanti-mouse IgG polyclonal (pAb) beads (FIG. 36A) and two batches ofanti-human IgG pAb beads (FIGS. 36B-36C) were prepared for performing aone-bead assay. FIGS. 36A-36C show that all batches of beads showedclustering when 10 μg/ml of mouse or human IgG were present. Each batchis shown against a no cell (NC) control.

FIG. 37 depicts images from tests performed using a one-bead assay asdescribed herein, used to assess mouse IgG secreting single hybridomacells in PODS.

FIG. 38 depicts images from tests performed using a two-bead assay asdescribed herein, used to assess antigen-specific antibody secretingsingle hybridoma cells in PODS.

FIG. 39A depicts 4× objective microscope images from the tests conductedusing the HB-123 cell line, wherein no clustering was expected.

FIGS. 39B-39D depict 10× objective microscope images at time=0, t=1hour, and t=3 hours, showing that clustering occurred starting from t=1hour.

FIG. 40A depicts a top plan view of a variation of a chamber arrangementin an open state. FIG. 40B depicts a top plan view of the chamberarrangement of FIG. 40A in a closed state. FIG. 40C depicts across-sectional side view of the chamber arrangement shown in FIG. 40B,taken along the line 40C:40C.

FIG. 41 depicts a top plan view of another variation of a chamberarrangement in an open state.

FIGS. 42A and 42B depict cross-sectional side views of another variationof a chamber arrangement in a partially closed state and a closed state,respectively.

FIG. 43A depicts a schematic illustration of a first binding partnercomplex for use in a one-bead or two-bead assay, using biotin coatedbeads and streptavidin conjugated antibodies. FIG. 43B depicts aschematic illustration of a first binding partner complex for use in aone-bead or two-bead assay, using streptavidin coated beads and biotinconjugated antibodies.

DETAILED DESCRIPTION

Non-limiting examples of various aspects and variations of the inventionare described herein and illustrated in the accompanying drawings.

Generally, described herein are exemplary variations of assay systemsand methods for processing samples. For example, such systems andmethods may process a large number of entities within the samplesubstantially in parallel, such as to enable rapid experimental analysisof the sample. Furthermore, the systems and methods described herein maybe used to process polydisperse entities of non-uniform size. Generally,the systems and methods described herein may facilitate measurements ofdiagnostic- and/or research-related events or sample characteristics,such as agglutination, colloidal stability, cell growth, cell surfaceprofiling, cell size profiling, and/or the profiling of concentration ofproteins, antibiotics, nucleotides, other analytes, and the like.Applications may include diagnostics, drug research, environmentalresearch, and the like.

PODS

As described in further detail below, the systems and methods may, forexample, process partitioned samples. For example, the systems andmethods may process suitable experimental dispersion, a type of which isalso referred to herein as Polydisperse Oblate Dispersion System(“PODS.”) A POD may include in its body any suitable experimentallyuseful content, such as bacterial or mammalian cells, DNA, RNA,nucleotides, proteins, enzymes, and/or any suitable chemical and/orbiological content for analysis. In other examples, a POD may includereagents that are used to confer signals to one or more image sensorssuch that the PODS may be processed by software to yield meaningfulchemical and/or biological information. PODS may, for example, be usedfor the early detection of molecules secreted from a mammalian cell,such as IgG from a hybridoma or B cell. Suitable reagents oragglutinates may include, for example, beads coated with gold, latex,cellulose, agarose, polystyrene, magnetic, and/or other materials boundto biologically active proteins or scaffolds (e.g., materials suitablefor ELISA kits and agglutination assays such as cell surface binding andcell agglutination assays). Additionally, in some variations (e.g., forsamples with cell cultures), a substance such as L-glutamine may beencapsulated in the PODS so as to help keep cells viable. Furthermore,in some variations, PODS may include hydrogels or a porous solid orpolymeric phase that serve as an anchor for a capture protein orantibody. A sandwich type assay can then be constructed with a samplethat is specific to the capture protein, and a second detection antibodythat is bound to a detection catalyst or enzyme such as Horse RadishPeroxidase, HRP. A darkening substrate such as PCIB can then be added.

For example, a POD could include any such bead having a size betweenabout 10 nm to about 50 μm, and coated with a biomarker (e.g.,antibody). As another example, a POD could include a bead having a sizebetween about 30 nm to about 50 μm. The degree of agglutinationresulting from self-aggregation of such reagents or agglutinates (whichmay be monodisperse or polydisperse) in the assay system describedherein may, for example, enable inference of protein and/or analyteconcentrations. Thus, analytes of interest include, but are not limitedto, various chemical and/or biological mixtures including buffers,cells, tissues, lysates, agglutinates, aggregate proteins, drugs,antibodies, nucleotides, dyes, and/or coated particles, etc. Exemplaryapplications of the systems and methods described herein are shown inFIG. 12 and described in further detail below.

In some variations, each POD may be considered a separate experiment,such that processing of multiple PODS enables the fast and efficientperformance of multiple experiments in parallel. Processing PODS mayinvolve, without limitation, analyzing one or more characteristics ofPODS, tracking location and/or predicting trajectory of PODS within thechamber, and/or manipulating PODS for sorting.

In some variations, a POD may include an aqueous phase that isstabilized and is transportable in a surrounding medium such as a liquidor other fluid (e.g., a non-aqueous solution containing a surfactant orlipid, or mixture thereof). In some variations, a POD being processed bythe assay device may be distinct from a droplet at least in part becausea POD is not spherical. For example, a processed POD might not bespherically symmetrical. The processed POD may be smaller in onedimension (e.g., in a dimension measured generally orthogonal to anelectrode surface as described below) than in another dimension (e.g.,oblate). For example, the processed POD may be generally flattened on atleast one side, similar to a generally hemi-spherical shape, or may begenerally flattened on at least two opposing sides, similar to adisk-like or “pancake” shape. As described in further detail below, aPOD that is flattened on at least one side may have increased surfacearea of contact with measurement electrodes in the assay device, suchthat electrode measurements may have reduced noise and generallyimproved signal quality. Additionally, as described in further detailbelow, a POD that is flattened on at least one side may bevolumetrically restricted so as to concentrate the POD contents into ashape approximating a two-dimensional focal plane of a camera, therebyimproving visibility of the POD contents by the camera. Furthermore, aPOD may be distinct from a droplet at least in part because multiplePODS being processed simultaneously by the assay device may bepolydisperse, in contrast to droplets which are conventionally thoughtof as being the same size (e.g., having monodisperse characteristics).

For example, a POD may be pressed into a flattened form (e.g., bymechanical compression between two plates, between opposing surfaces ofa chamber such as that described below, or other suitable mechanism), byincreasing surfactant concentration, or in any suitable manner.

The surrounding medium for the PODS may, for example, include anon-aqueous continuous phase. In some variations, the surrounding mediummay be fluorous. For example, the medium may include a fluorinated oilor other liquid (e.g., HFE 7500 available as Novec™ manufactured by 3M®or FC-40, available as Fluorinert™ manufactured by 3M). As anotherexample, the medium may include hydrocarbon oil. The medium may, in yetother variations, additionally or alternatively include PEG andfluoridated derivatives (e.g., derivatives of Krytox™ fluorinated oilsmanufactured by The Chemours Company, which may be polymerized orco-polymerized with PEG or other suitable glycol ethers), and mayinclude lipids or other phosphoric, carboxylated or amino-terminatedchains.

In some variations, a POD may have an overall density that is lower thanthe density of the surrounding medium, such that aqueous PODS within themedium are more buoyant and tend to rise within the surrounding medium.For example, the surrounding medium may include a fluid denser thanwater, such as HFE-7500 and/or FC-40, which may be mixed with co-blockpolyethylene glycol/Krytox™ polymer. In other variations, a POD may havean overall density that is higher than the density of the surroundingmedium such that aqueous PODS within the medium are less buoyant tend tosink within the surrounding medium. For example, the surrounding mediummay include a fluid less dense than water, such as hexadecane and aphospholipid bilayer. In yet other variations, a POD and its surroundingmedium may have substantially similar or equal densities. It should beunderstood that various combinations of relative densities of PODS andthe surrounding medium may provide varying levels of buoyancy of thePODS within the surrounding medium (e.g., a set of PODS within aparticular medium may include some PODS that tend to rise and some PODSthat tend to sink). For example, relative buoyancy of the PODS may bebeneficial in some applications to leverage gravity in the sorting ofPODS. However, the POD may be surrounded by any suitable medium.

One or more PODS may be introduced in combination with a suitablesurrounding medium as an emulsion into an assay device and processed asdescribed herein. In some variations, mixing to create PODS may occuroutside of the assay device (e.g. adjacent an external side of an inletof the device prior to introduction into the device), while in othervariations such mixing may additionally or alternatively occur insidethe assay device. For example, PODS may be generated by agitating twosolutions including a biological reagent and a fluorinated liquid.Furthermore, larger PODS may be transformed into smaller PODS (e.g., byinteraction with spacers in the assay device as described below, orinteraction with any other suitable device feature) to control or adjustpolydispersity among the PODS.

The assay devices and methods may be used to process polydisperse sampleentities. For example, various aspects of the devices and methodsdescribed herein may enable substantially simultaneous processing ofPODS of different sizes, in contrast to conventional systems whichrequire samples to be monodisperse. In some variations, the assaydevices and methods described herein may simultaneously process sampleentities having at least 5%, at least 10%, at least 25%, or at least 50%variance in size (e.g., POD diameter, POD circumference, POD surfacearea, POD volume, etc.). The ability to handle polydisperse samples may,for example, provide sample analysis that is simpler and more efficient(e.g., by not requiring the sample entities to be sorted by size in aseparate, time-consuming process before introducing them into an assaydevice).

Exemplary applications of the assay devices and methods described hereininclude processing PODS to measure analyte concentration, measure celldivision, measure morphology, size, and/or number of cells or particleswithin a POD or other sample entity, measure relative sizes of cells(and/or agglutinates) and the PODS within which they are contained(e.g., ratio between circumference of a POD and the circumference of acell within the pod), and the like. For example, the devices and methodsmay be used for pathology, oncology, determining white or red blood cellcounts, etc. Furthermore, the assay devices and methods described hereinmay be used to perform any of a wide variety of agglutination tests.

Assay System for Processing a Sample

Generally, as shown in the schematic of FIG. 1A, in some variations, anassay system 100 for processing a sample includes a chamber 120 havingat least one inlet 122 and at least one outlet 124, wherein the chamberis configured to accommodate flow of the sample from the at least oneinlet toward the at least one outlet, and an imager array 140 configuredto image the flow of the sample in the chamber 120. The imager array 140may include at least one lensless image sensor configurable opposite atleast one light source 130. In some variations, the assay system 100 mayinclude a fluidic control system with one or more pumps, valves, and/orfluid sensors to manipulate flow of the sample. The system 100 mayfurther include an electronics system 160 (e.g., PCBA with one or moreprocessors, etc.) configured to control and/or receive signals fromother components of the assay system 100, as further described below. Insome variations, the electronics system 160 may further include one ormore communication components (e.g., Bluetooth, WiFi, etc.) configuredto communicate data (e.g., image data) to a network 170 for analysis byone or more remote processors 180. Additionally or alternatively, atleast some of the data may be analyzed by one or more processors locatedin the electronics system 160.

FIG. 1B depicts a schematic of an exemplary variation of a system 100for processing a sample including a chamber 120 configured to receive asample (e.g., emulsion) from a reservoir 116 coupled to an inlet of thechamber 120. The chamber 120 may be arranged between one or more lightsources 130 and an imager array such that the imager array may produceoptical shadow images of the sample within the chamber 120. The imagesmay be analyzed using techniques such as those described herein, thesample may be processed (e.g., characterized and output into one or morewaste containers such as a reservoir 156 and/or other receptacle 156′(e.g., Eppendorf tube). Furthermore, the system 100 may include arobotic or automated pipette 190 for drawing portions of the sample thatmay be of interest for further analysis or other processing.

Chamber Arrangement

As described above, the assay system may include a chamber having atleast one inlet and at least outlet, and may be configured toaccommodate flow of the sample from the at least one inlet toward the atleast one outlet. Generally, the chamber may be configured toaccommodate a two-dimensional flow of the sample, such that PODS (orother entities in the sample) may circulate within the volume of thechamber (e.g., in multi-directional flow). For example, the chamber mayinclude a generally rectangular volume. In some variations, the chambermay be defined at least partially by a first structure and a secondstructure opposing the first structure, where each of the first andsecond structure has at least a portion that is optically transparent.In some variations, the chamber may be implemented at least in part on aflexible printed circuit board (“flex” circuit).

Furthermore, at least one light source may be positioned on one side ofthe sample flow in the chamber, and an imager array including at leastone lensless image sensor may be positioned on the other side of thesample flow (opposite the light source) in the chamber. In such anarrangement, the imager array may be configured to generate “shadowimages,” or images through shadowgraphy, of chamber contents that arebacklit by the at least one light source. Information (e.g., chemicaland/or biological information) about samples may be derived from suchshadow images of the samples.

In some variations, the assay device may additionally or alternativelyinclude one or more electrodes configured to measure electroniccharacteristics of samples (e.g., perform impedance measurements thatmay be correlated to chemical and/or biological information about thesamples, for example) and/or generate electrical fields to enabledielectrophoresis. For example, the chamber may include electrodessimilar to those described in U.S. patent application Ser. No.15/986,416 which is hereby incorporated in its entirety by thisreference. Additional examples of such electrodes are described infurther detail below, with respect to exemplary variations of chamberarrangements.

Generally, as shown in in the cross-sectional view schematic of FIG. 2A,a chamber arrangement may include a chamber 200 having a first structure210 and a second structure 212, where the first and second structuresinclude an optically transparent material and are offset from each otherto form a gap 214 or at least partially defining a chamber volume.Spacing between the first structure 210 and the second structure 212may, in some variations, be supported or enforced by one or more spacers216 as further described herein. Thickness of spacers may be determinedto, for example, adjust chamber height and/or operational parameterssuch as emulsion stability, POD flow rate, etc. In some variations,chamber height may be at least part based on the kind of PODS or sampledesired to be analyzed. Suitable chamber heights may range, for example,between about 0.1 μm to about 200 μm. For example, some PODS may includecells that may be best analyzed using a chamber having a taller heightsuch as 25-30 μm, while some PODS may include proteins that may be bestanalyzed using a chamber having a shorter height such as less than 1 μm.

The first structure 210 and the second structure 212 may includemulti-layer stackups formed with semiconductor planar processingtechniques (e.g., adding material on a substrate with deposition,sputtering, plating, and/or immersion processes, subtracting material tointroduce patterning such as with photolithography or other etchingprocesses, or laser-defined imaging processes, etc.). A layer may be acontinuous structure (e.g., a nonpatterned thin film) or a discontinuousstructure (e.g., a patterned thin film with cutouts, gaps, etc.). Byutilizing such planar processing techniques, the structures forming thechamber may be dimensionally scaled at low cost. Scalability across aplane enables the assay device to image or detect many PODSsimultaneously, thereby increasing analysis throughput, or the totalnumber of events (e.g., PODS, or reactions within PODS, etc.) that maybe detected over a period of time. Furthermore, these manufacturingtechniques enable precise control of chamber height, shape, andfootprint area, thereby allowing for flexibility in customizing theoverall assay device for a wide range of applications (e.g., sampletypes with different POD sizes, for example).

A light source 230 may be positioned on one side of the chamber and beconfigured to emit light toward the gap 214. An imager array 240 with alensless image sensor (e.g., CMOS imager) may be positioned on the otherside of the chamber, opposite the light source 230, and configured toimage the region of the gap 214. Specifically, the lensless image sensormay be placed directly on the chamber (or alternatively used to directlyform the boundary of the chamber), without an objective lens or otheroptical focusing lenses in the line of sight between the lensless imagesensor and the chamber. The first structure 210 and the second structure212 may include an optically transparent material, such that light fromthe light source 230 may pass through an optically transparent portionof the first structure 210, travel across the gap 214, pass through anoptically transparent portion of the second structure 212, and beincident on the imager array 240.

A sample may flow through the chamber 200 in the gap 214, as representedin FIG. 2A as a POD passing through gap 214. For purposes ofillustration, the POD can include an analyte such as an agglutinate, asshown in FIG. 2A, though it should be understood that a POD can includeother kinds of analytes (or no analyte). Light from the light source 230may be emitted toward the chamber (and toward the POD within thechamber) and interact with the POD and its contents when the POD is inthe chamber. The imager array 240 may be configured to detect and imagethe optical phenomena resulting from these interactions, including, forexample, shadows, absorbance or emission spectra (e.g., fluorescence),extinction coefficient, light scattering, etc.

For example, FIG. 2A illustrates a system in which the imager array 240is configured to generate shadow images of the sample flow in thechamber. The light source 230 may be configured to emit light (e.g.,visible light) toward the sample flow. As shown in FIG. 2A, some lightrays (e.g., light rays “A”) may enter the chamber and pass through theaqueous portion of the POD relatively undisturbed, which causes theaqueous portion of the POD to be imaged by the imager array 240 as abright, backlit region (e.g., region IA in FIG. 2B). Some light rays(e.g., light rays “B”) may enter the chamber and be scattered orreflected due to the agglutinate (or other analyte(s)) in the POD, whichcauses the agglutinate (or other analyte(s)) to be imaged by the imagerarray 240 as a somewhat darkened, indefinite or “fuzzy” region (e.g.,region I_(B) in FIG. 2B). In some variations, information about the PODand its contents, such as size, shape, and/or density of theagglutinate, may be determined based at least in part on the darkened,indefinite region of the image (e.g., based on size, shape, pixelintensity, etc. of the region). Furthermore, some light rays (e.g.,light rays “C”) may enter the chamber and undergo diffraction at the PODboundary, which causes the POD boundary to be imaged as a dark, shadowedborder region (e.g., I_(C) in FIG. 2B). In some variations, the overallshape and/or size of the POD may be determined based at least in part onthe border region (e.g., shape, size, pixel intensity, etc. of theborder region). Accordingly, one or more lensless image sensors in theimager array 240 may be configured to generate “shadow images” of thebacklit contents of the chamber. Chemical and/or biological propertiesmay be derived from these shadow images.

FIG. 9 is an exemplary shadow image of sample flow in a chamber such asthat shown in FIG. 2A. The shadow image is the result of processing araw shadow image taken by a lensless CMOS image sensor adjacent thechamber and opposite a light source that provides backlighting of thesample flow in the chamber. The sample flow includes multiplepolydisperse PODS passing through the chamber. Some of these PODSinclude a bead about 22 μm in diameter, which is approximately the sizeof a circulating tumor cell and may be coupled to an antibody. Thus, thebead has an analyte that may be imaged (or is otherwise representativeof another analyte that can be similarly imaged), if present in the POD.For example, a POD that includes a bead (e.g., latex, polystyrene,magnetic material, gold, etc.) coated with a biomarker may have avisually distinct pattern when the contents of the POD also include anentity that reacts or binds to the biomarker (e.g., epitope, antigen, orother marker). Such a visually and/or quantifiably distinct pattern (orchange in pattern) may be used to quantitate the biomarker. As shown inthe shadow image of FIG. 9, the size and shape of the POD areidentifiable and measurable based on the appearance of darkened patternswithin the PODS. Additionally, characteristics of beads in certain PODSare identifiable, such as presence of beads, size, shape, etc.Furthermore, by analyzing the appearance of the PODS and their contentsover time across multiple shadow images, dynamic characteristics (e.g.,movement and/or change in shape or size of POD contents) can be analyzedto provide additional information about the chemical and/or biologicalnature of the PODS.

FIG. 10 is another exemplary shadow image of sample flow in a chambersuch as that shown in FIG. 2A. The sample flow includes multiplepolydisperse PODS passing through the chamber. Some of these PODS (e.g.,PODS 1010), may contain one or more red blood cells, while some PODS(e.g., PODS 1020) may be “empty” in that they lack red blood cells orother analytes. As shown in the shadow image of FIG. 9, the size andshape of the POD are identifiable and measurable based on the appearanceof darkened lines outlining the PODS. Additionally, characteristics ofthe red blood cells in certain PODS are identifiable (e.g., number ofred blood cells, etc.). Furthermore, similar to that described abovewith respect to FIG. 9, by analyzing the appearance of the PODS andtheir contents over time across multiple shadow images, dynamiccharacteristics (e.g., movement and/or change in shape or size of PODcontents) can be analyzed to provide additional information about thechemical and/or biological nature of the PODS.

FIGS. 11A and 11B illustrate another exemplary variation in which theimager array is configured to obtain fluorescent images of the sampleflow in the chamber. FIGS. 11A and 11B illustrate a chamber arrangementhaving a first structure 1110, a second structure 1112 offset with a gap1114 as previously described and similar to the chamber arrangementdescribed above with reference to FIG. 2A, except as described below. Asshown in FIG. 11A, the light source 1130 may be configured to emit light1132 suitable for inducing fluorescence or other emission spectra towardthe sample flow. The emitted light 1132 may, for example, includeultraviolet light (UV). At least some PODS in the sample flow mayinclude a bead or biological sample 1102 or other substance configuredto absorb the emitted light and emit light in response (e.g., of adifferent wavelength). For example, as shown in FIG. 11B, at least someemitted light may be absorbed by a POD or contents therein, which may inturn emit fluorescence or other light emission 1134. The emittedfluorescence may be imaged as a fluorescent image by at least a portionof the image sensors in the imager array 1140. Chemical and/orbiological properties may be derived from these fluorescent images(e.g., based on wavelength of emitted light, intensity of emitted light,etc.).

Furthermore, although the chamber arrangement of FIGS. 11A and 11Bdepict an imager array 1140 that is opposite the light source 1130emitting light for inducing fluorescence, it should be understood thatin other variations, the imager array 1140 may be located in anysuitable location proximate the light source 1130 so as to capturefluorescence or other emission spectra from the sample flow. Forexample, at least a portion of the imager array 1140 and at least aportion of the light source 1130 may be orthogonal to each other (e.g.,one on a side wall of the chamber 1100, the other on an upper structureor lower structure of the chamber 1100). As another example,additionally or alternative, at least a portion of the imager array 1140and at least a portion of the light source 1130 may be adjacent to eachother (e.g., on the same surface such as on the upper structure or lowerstructure, in an alternating or other distributed pattern).

Lensless imaging may provide several advantages compared to conventionaloptical systems with lenses. For example, lensless image sensors mayprovide high resolution imaging over a large field of view. This mayenable the imager array to successfully image a high number of PODS(e.g., over 100, or over 200) in the chamber in a single image frame.Furthermore, because lensless image sensors do not require focusing,there may be less need for precise optical alignment and positioning ofoptical components, thereby easing manufacturing processes and reducingburden on the user and/or software to adjust the focus of the imagerarray. The absence of lenses may also alleviate challenges with focalgradients that are common in lenses, and lowers overall part count andcost of the assay device. Accordingly, the incorporation of lenslessimage sensors in chamber arrangements (such as those described herein)may further enable dimensional scalability at low cost.

The arrangement of the chamber, the one or more light sources, and theimager array may be constructed in various suitable manners. Forexample, FIG. 3 shows an exemplary variation of an assay system 300 forprocessing a sample. Generally, assay system 300 may include componentssimilar to assay system 100 described above and shown in FIG. 1. Asshown in FIG. 3, assay system 300 further includes one or more supports,including a base 380 and a light source post 334 coupled to the base380. The base 380 may include a plate or other suitable stable surfacefor receiving a chamber 320, an imager array (not shown) underneath thechamber 320 and/or at least part of an electronics system 360. Forexample, the base 380 may include at least one recess shaped tocomplementarily receive the chamber 320, imager array, and/orelectronics system 360. The chamber 320, imager array, and/orelectronics system 360 may be coupled to the base 380 with fasteners,epoxy, interlocking mating features, and/or other suitable features. Insome variations, the base 380 may be secured to a desktop, tabletop, orother suitable surface directly or indirectly (e.g., via a base mount382 such as a secondary plate).

A light source post 334 may be mounted to the base 380 (e.g., withfasteners or interlocking features, etc.). In some variations, the lightsource post 334 may be vertical and mounted orthogonally to the base380. A light source housing 332 may house a light source (e.g., LED, orcoherent light sources such as a laser, or other suitable light source)and may be coupled to the light source post 334 (e.g., via a clamp orpin mechanism, etc.) such that that the light source is positioned overthe chamber 320 on the base 380. The light source housing 332 may beadjustably coupled to the light source post 334 so as to enableadjustment of the relative positions of the light source and the chamber320. For example, an adjustment knob 336 may be turned to loosen a clampthat couples the light source housing 332 to the light source post 334,such that the light source housing 332 may be adjusted vertically alongthe light source post 334. Upon the light source housing 332 beingpositioned in a desired location, the adjustment knob 336 may betightened to secure the position of the light source housing 332 on thelight source post 334. In other variations, other suitable mechanismsmay enable adjustment of the light source housing (e.g., threadedattachments, one or more pins insertable in holes located at discreteheights, etc.). Furthermore, it should be understood that in somevariations, the chamber 320 location may additionally or alternativelybe adjusted (e.g., by moving the location of the base 380) relative tothe light source. In some variations, the relative locations of thelight source and the chamber are such that the light emitted from thelight source are substantially collimated when incident on and enteringthe chamber. In one exemplary variation, the light source housed in thelight source housing 332 may include one or more white light LEDspositioned at a distance of about six inches above the chamber mountedin the base 380.

An exemplary variation of a chamber arrangement is shown in FIGS. 4A-4D.As best shown in FIG. 4B, the chamber arrangement includes a chamber 400with a first, upper structure 410 and a second, lower structure 430. Forexample, as shown in FIGS. 4A and 4C, the upper structure 410 mayinclude a laminate composite including an optically transparent layer412, a patterned structural layer 416 (e.g., copper or other suitablemetal), and an adhesive layer 414 (e.g., acrylic adhesive) for bondingthe optically transparent layer 412 and the structural layer 416together. The lower structure 430 may include a laminate compositeincluding an optically transparent layer 434, an upper patternedstructural layer 432, a lower patterned structural layer 436, andadhesive layers 433 and 435 for bonding the optically transparent layer434 to the upper and lower patterned structural layers, respectively.The optically transparent layer, structural layers, and adhesive layersin the lower structure 430 may be made of similar materials as the upperstructure 410. Exemplary materials for at least part of one of theabove-described optically transparent layers include polyimide and glass(e.g., flexible Willow® glass manufactured by Corning®, or othersuitable glass material), other suitable optically transparentsubstrates, or any combination thereof.

As shown in FIG. 4A, one or more light sources 490 may be located on oneside of the chamber volume, and an imager array 492 may be located onanother side of the chamber volume opposing the one or more lightsources 490. For example, one or more light sources 490 may be locatedabove the upper structure 410 and directed to emit light toward thechamber volume. In some variations, a light source 490 may be an LED orother suitable light source embedded or placed within layers of theupper structure 410, while in other variations a light source 490 may belocated external to the upper structure 410. An imager array 492 may belocated below the lower structure 440 and directed to image the chambervolume. In some variations, the imagery array 492 may include one ormore lensless CMOS image sensors. Alternatively, one or more lightsources 490 may be located below the lower structure 430 and the imagerarray 492 may be located above the upper structure. Furthermore,although the chamber 400 is shown and described with the structures 410and 430 being upper and lower structures, it should be understood thatthe orientation of chamber, light sources, and imager array may bedifferent (e.g., the orientation may be rotated 90 degrees or 180degrees from that shown in FIG. 4A).

The upper structure 410 and the lower structure 430 may be joinedtogether, such that the upper structure 410 provides an upper surface ofthe chamber 400 and the lower structure 430 provides a lower surface ofthe chamber 400, as shown in the orientation of FIG. 4A. For example,the upper structure 410 and lower structure 430 may be bonded at leastin part by an intervening adhesive layer 420, where the adhesive layer420 may have a channel cutout 422 providing a central empty space forthe chamber volume between the upper structure 410 and the lowerstructure 430. Registration features, such as alignment holes 470 ineach of the upper structure 410 and lower structure 430, may facilitatealignment of the structures to form the chamber.

One or more spacers may be located in the chamber volume to support thespacing between the upper structure 410 and the lower structure 430and/or facilitate coupling of the upper structure 410 and the lowerstructure 430. As shown in the top plan view of FIG. 4D and thecross-sectional view of FIG. 4A, one or more boundary spacers 426 mayform side walls of the chamber volume. For example, a boundary spacer426 may be generally oval-shaped or rectangular-shaped (e.g., mayinclude linear sides located on left and right sides of the chamber asshown in FIG. 4D). Furthermore, one or more spacer posts 424 may bearranged within the chamber volume. The spacer posts 424 may, in somevariations, provide columnar support to enforce spacing between theupper and lower structures of the chamber. In some applications, thespacer posts 424 may additionally function to break up aggregated PODS,induce turbulence in sample flow, and/or other affect flow of the samplein the chamber. The spacer posts 424 may be distributed in a regulararray (e.g., rectangular array as shown in FIG. 4D. staggered array asshown in FIG. 24A), or alternatively in an irregular array or othersuitable pattern.

Although the boundary spacers 246 are depicted in FIG. 4D as elongated,linear strips, it should be understood that other shapes (e.g., wavystrips, irregular lengths) may be suitable. Furthermore, the boundaryspacers 246 may be intermittently placed on the sides of the chamber,such as to accommodate additional sample inlets and/or outlets for thechamber between intermittent boundary spacers 246. Similarly, althoughthe spacer posts 424 are depicted in FIG. 4A as having squarecross-sections, it should be understood that in other variations, thespacer posts 424 may have other suitable cross-sections, such ascircular or triangular.

In some variations, the spacers 424 and/or 426 may be formed from thepatterned structural layers of the upper structure 410 and lowerstructure 430. For example, the patterned structural layer 416 of theupper structure 410 may adjoin the patterned structural layer 432 of thelower structure 430 such that the structural layers in combination formthe spacers 424 and/or 426. In some variations, the structural layers416 and 432 may be equal in thickness so as to each provide half of theheight of the spacers. In other variations, the structural layers 416and 432 may have different thicknesses (e.g., the structural layer 416may be thicker or thinner than the structural layer 432). Alternatively,in some variations, the spacers 424 and/or 426 may be formed from anycombination of layered structures. Furthermore, additionally oralternatively, non-layered components (e.g., beads) may provide spacingbetween the upper and lower surfaces of the chamber 400.

In some variations, as shown in the detailed view of FIG. 5, the heightof the spacers relative to the thickness of the intervening adhesivelayer 420 between the upper structure 410 and the lower structure 430may be selected to introduce a “stretched drum” effect on the upperstructure 410 to enhance the coupling between the upper and lowerstructures and/or compress the patterned layers forming the spacers. Forexample, the height of the spacers 424 and/or 426 may be slightlygreater than the thickness of the adhesive layer 420, such that theupper structure 410 is generally urged downward toward the lowerstructure 430 at the border of the chamber volume. This “stretched drum”effect may, for example, help compress the upper structure 410 and thelower structure 430 together and against the adhesive layer 420, so asto form a fluidic seal for the chamber volume. In some variations, theratio between the height of the spacers and the thickness of theadhesive layer 420 may be between about 2 and about 4, or about 3.

Furthermore, as shown in FIG. 4A, at least some of the spacers mayinclude a via 460 providing a passageway through the layers of the lowerstructure 430. The vias 460 may provide a passageway for an anchormaterial to bond the upper structure 410 and lower structure 420together. For example, as shown in FIG. 4C, an anchor material 462 maybe introduced into the via 460 to bond directly to the upper structure410 and the lower structure 420, thereby joining the structures. In somevariations, the anchor material may be solder or a polymer adhesive. Inan exemplary method, the anchor material may be introduced into a viawith the use of a patterned stencil having an opening aligned with thevia (or multiple openings aligned with multiple vias). After placing thestencil over the via (e.g., on the underside of the lower structure430), anchor material may be scraped over the stencil to force theanchor material into the via. Excess anchor material will stay on thestencil and be removed by removing the stencil and/or being wiped offthe stencil, thereby leaving only anchor material in the via. Suchanchoring may be used in addition to, or as an alternative to, theintervening adhesive layer 420 described above for bonding the upperstructure 410 and the lower structure 420.

In addition to the upper and lower structures 410 and 430 describedabove, in some variations the chamber arrangement may further include astiffener layer 450 and a stiffener adhesive layer 440 for bonding thestiffener layer 450 to the rest of the chamber 400 (e.g., to theunderside of the lower structure 430). The stiffener layer 450 mayprovide structural support for manipulating and/or connecting components(e.g., port for a chamber inlet and/or chamber outlet) to the chamber400. Like the first structure 410 and the second structure 430, thestiffener layer 450 and adhesive layer 440 may include referencefeatures (e.g. holes 470) to enable alignments with the rest of thestackup layers. As shown in FIG. 4B, the stiffener layer 450 may includea channel cutout 456, and the adhesive layer 440 may include a channelcutout 442, where the channel cutouts 456 and 442 provide space for theimager array 492 in the fully assembled stackup. An additionalstructural layer 454 (e.g., copper or other metal) may also be bonded tothe stiffener layer 450 for further structural support.

In an exemplary embodiment of the chamber arrangement shown in FIG. 4A,the upper structure 410 can include a one-sided copper-clad laminatedcomposite (such as laminate composite LF8510 manufactured by DuPont™)including a 1 mm thick polyimide film that is bonded on one side facingthe chamber volume to a 0.5 oz (18 μm thick) copper foil with an acrylicadhesive. The copper foil is patterned to form an oval-shaped boundaryspacer and a plurality of partial spacer posts arranged in a rectangulargrid inside the oval-shaped boundary. The lower structure 430 caninclude a dual-sided copper-clad laminated composite (such as laminatecomposite LF7005 manufactured by DuPont™), where an upper copper sidefacing the chamber volume is 0.5 oz (18 μm thick) copper foil and alower copper side facing away from the chamber volume is 1 (36 μm thick)copper foil. The upper and lower structures of the chamber are flexible.A 12.5 μm thick polymer adhesive layer with an oval-shaped channelcutout is located outside the perimeter of the boundary spacer to bondthe upper and lower structures of the chamber together. Both the upperand lower copper sides of the lower structure 430 are patterned to forma plurality of partial spacer posts, such that the facing patternedcopper foil layers combine to form a plurality of spacer posts with 100μm diameter circular vias. Solder in the vias, in combination with thepolymer adhesive, anchor the upper and lower structures of the chambertogether. Furthermore, the lower structure 430 may be strengthened suchas with a stiffener backing. For example, a FR4 stiffener with copperfoil on one side may be bonded to the underside of the lower structure430, such as with a suitable adhesive (e.g., 1 mm thick polymeradhesive).

Another exemplary variation of a chamber arrangement is shown in FIGS.7A and 7B. Similar to the chamber arrangement described above withrespect to FIGS. 4A-4D, a chamber arrangement can include a chamber 700including an upper structure 710 and a lower structure 730 offset fromthe upper structure 710. As shown in FIG. 7A, one or more light sources750 may be located on one side of the chamber volume, and an imagerarray 740 may be located on another side of the chamber volume opposingthe one or more light sources 750. For example, one or more lightsources 750 may be located above the upper structure 710 and directed toemit light toward the chamber volume (e.g. gap 702). In some variations,a light source 750 may be an LED or other suitable light source embeddedor placed within layers of the upper structure 710, while in othervariations a light source 750 may be located external to the upperstructure 710. An imager array 740 may be located below the lowerstructure 740 and directed to image the chamber volume. In somevariations, the imagery array 740 may include one or more lensless CMOSimage sensors. Furthermore, although the chamber 700 is shown anddescribed with the structures 710 and 730 being upper and lowerstructures, it should be understood that the orientation of chamber,light sources, and imager array may be different (e.g., the orientationmay be rotated 90 degrees or 180 degrees from that shown in FIG. 7A).

Similar to the chamber arrangements described above, the upper structure710 and the lower structure 730 may be offset by a gap 702. The gap 702may be supported or enforced by one or more spacers 720. Similar to theupper structure in the chamber arrangement shown in FIG. 4A, the upperstructure 710 may include a multi-layer stackup. For example, the upperstructure 710 may include a laminated stackup of an opticallytransparent material and adhesive (e.g., polyimide and an acrylicadhesive, or other suitable materials). Furthermore, the upper structure710 may include material forming one or more spacers 720 (e.g., boundaryspacers forming at least a portion of the boundary of the chamber,spacer posts located within the chamber volume). For example, a spacer720 may include a plurality of bonded layers including copper 722, gold726, and/or other surface plating 724 (e.g., electroless nickelimmersion gold, or ENIG) that collectively form the spacer 720. Althoughthe spacers 720 are depicted as including layers of material in theupper structure 710, it should be understood that the layers of materialforming a spacer 720 may additionally or alternatively include layers ofmaterial in the lower structure 730.

As shown in FIG. 7B, the lower structure 730 may include may include anoptically transparent material (e.g., polyimide) and one or more vias732. A copper layer 736 may line vias 732. Similar to the vias in thechamber arrangement described above with reference to FIG. 4A, the vias732 may accommodate anchor material 734 for bonding the upper structure710 to the lower structure 730. For example, solder, adhesive, oranother suitable anchor material may be deposited in one or more vias732, and adjoin the upper structure 710 (e.g., the material forming thespacer 720) with the lower structure 730, thereby securing the upper andlower structures together.

As shown in FIG. 7B, the chamber 700 may include electrodes 760 that areexposed to the chamber volume and interact with sample flowing withinthe chamber. The electrodes 760 may be generally arranged as an array,for example. In some variations, at least some electrodes may beconfigured to measure electronic properties (e.g., impedance) ofsamples. Additionally or alternatively, at least some electrodes may beconfigured to generate an electrical field to enable dielectrophoresis.As shown in FIG. 7B, the electrodes 760 may be connected to conductivetraces 762 (e.g., patterned copper). Such traces may be patterned toextend on a planar surface (e.g. into or out of the page relative to thecross-sectional view of FIG. 7B) and enable signals to pass to and fromthe electrodes 760. In other words, the electrodes 760 and theirpatterned electrical ads and traces may be directly integrated andelectrically connected within the stackup, and may further be connectedto electronics components (e.g., controller, processor, etc.) onboardthe assay device or on one or more external computing devices.

In an exemplary embodiment of the variation shown in FIGS. 7A and 7B, achamber arrangement may include an upper structure that includes alaminated structure of alternating polyimide and adhesive layers. Theupper structure can include an upper polyimide layer 712 having athickness of about 50 μm, an intervening adhesive layer 714 (such asacrylic adhesive LF0200 manufactured by DuPont™) having a thickness ofabout 50 μm, and a lower polyimide layer 716 having a thickness of about25 μm. The chamber arrangement further includes a lower structure thatincludes a polyimide layer of about 25 μm. The upper and lowerstructures are offset from each other and separated by a set of spacers,thereby forming a chamber volume through which fluid can flow. A seriesof white LEDs are arranged to emit white light through the upperstructure toward the chamber volume, and an imager array includinglensless image sensors (e.g., one or more color CMOS sensors such asOV5640 manufactured by Omnivision Technologies Inc. with lenses removed)is arranged opposite the LEDs to image the contents of the chambervolume. The upper structure further includes a set of patterned copper,ENIG, and gold layers that form the spacers between the upper and lowerstructures. Lateral spacing of the spacers may be about 450 μm. Vias inthe lower structure may receive solder material that extends through thelower structure to these spacers, thereby bonding the lower and upperstructures together. Additionally, the solder may be manipulated to flowout laterally on the underside of the lower structure to form aflange-type feature, thereby further securing the lower and upperstructures together. Furthermore, an array of exposed electrodes may bepatterned into the side of the upper structure facing the chamber volumeso as to interact with contents of the chamber volume. The electrodesmay be made of indium tin oxide (ITO) or other suitable thin filmconductor, and may be connected to copper conductive pads and tracespatterned throughout the upper structure.

Another exemplary variation of a chamber arrangement is shown in FIGS.8A and 8B. The chamber arrangement is similar to those described abovewith respect to FIGS. 4A-4D and FIGS. 7A and 7B, with additional detailsdescribed below. For example, a chamber arrangement can include achamber 800 including an upper structure 810 and a lower structure 830offset from the upper structure 810 by a gap 802. The gap 802 may beenforced or supported by one or more spacer posts 820 and/or boundaryspacers 821.

In the chamber arrangement shown in FIGS. 8A and 8B, one or more lightsources 850 and the imager array 840 are embedded, or integrated, withinthe upper and lower structures of the chamber 800. Specifically, theupper structure 810 of the chamber 800 may include one or more layers ofan optically transparent material (e.g., upper layer 812 and lower layer816), laminated with an adhesive layer 814. The upper structure 810 mayfurther include one or more lighting layers 818 within which at leastone light source 850 and its conductive pads and/or traces 852 arelocated. The lighting layers may include, for example, stiffenermaterial such as FR4 stiffener. Similarly, the lower structure 830 ofthe chamber 800 may include one or more layers of an opticallytransparent material (e.g., layer 830), and one or more imager layers832 within which the imager array 840 and its conductive pads and/ortraces 842 are located. Conductive traces 852 and 842 may pass to powersource(s) and controller(s) for operating the light source 850 andpassing signals to and from the imager array 840. For example,conductive traces may pass to an electronics region 870 of the assaydevice (e.g., disposed on an external surface of the upper structure,outside of the chamber 800) as shown in FIG. 8B, where the electronicsregion 870 may include electronic components relating to operation ofthe assay device. Suitable components include integrated circuits andpassive components, power sources, controllers, processors, datatransmitters, and/or memory, etc. In some variations, electroniccomponents may process signals and communicate results to externalcomputing devices and/or other peripheral devices.

Additionally, an electrode array including electrodes 860 may bepatterned onto the upper structure 810 and/or lower structure 830similar to that described above. Conductive pads and traces 862 may befurther patterned into the structures and passed to the electronicsregion 870, or another suitable region with electrode controlcomponents.

Similar to the chamber arrangements described above, vias passingthrough parts of the stackup may receive an anchor material to couplethe upper structure 810 and the lower structure 830. For example, theupper structure 810 may further include additional layers (e.g., copper)that are patterned to form spacer posts 820 and/or boundary spacers 821.The spacer posts 820 and/or boundary spacers 821 may include vias thatreceive a solder material extending through the layers 812-816 of theupper structure 810 and through the spacer materials, and bonding to thelower structure 830, thereby coupling the upper and lower structures.

In some variations, the chamber arrangement may comprise a substratethat includes and upper structure and a lower structure on differentsubstrate portions, the substrate may folded such that the upperstructure and the lower structure oppose each other. The foldedsubstrate may be sealed to form a chamber between the upper structureand lower structure. Exemplary variations of such chamber arrangementsare depicted by schematic illustrations in FIGS. 40A-40C, 41, and42A-42B.

The chamber arrangement variations shown in FIGS. 40A-40E are similar tothose described above, such as those described with respect to FIGS. 2Aand 2B, FIGS. 7A and 7B, and FIGS. 8A and 8B, except with respect to theadditional details described below. For example, a chamber arrangementcan be a single piece fluid device which includes a chamber 4000including a first structure 4010 and a second structure 4012 that areintegrally formed (e.g., cut out or otherwise formed from the samesubstrate such as a flex film). The structures may be formed (e.g.,deposited with thin film techniques, etc.) on different substrateportions connected by one or more flexible hinges 4029. The single piecefluid device may be foldable to create a sealed fluid chamber betweenthe first and second surfaces, wherein the sealing may be accomplishedafter the folding by the application of small external clamping pressureby screws, spring clamps, toggle clamps, or any other suitablemechanical means (not shown). Additionally or alternatively, the firstand second surfaces may be sealed by heating, adhesives, or any othersuitable sealing process.

FIG. 40A shows a top plan view of an exemplary embodiment of the singlepiece fluid device in an unfolded state, showing the first structure4010, and spacer posts 4024 provided in the channel 4022 of the secondstructure 4012. The first structure 4010 may act as a cover when thesingle piece fluid device is in a folded state (shown in FIG. 40E), andthe spacer posts 4024 may be arranged in an array such as a rectangulararray as shown and described when referring to FIG. 4A, for example, orin a staggered array as shown and described when referring to FIG. 23D,for example. Again, the spacer posts 4024 arranged in a staggered arraymay be configured to perform particle separation via deterministiclateral displacement (DLD). The single piece fluid device may beprovided with alignment holes 4089, which may assist in alignment of thesubstrate portions upon folding (e.g., to help guide relative positionsof the first and second structures), the placement of the device intoany suitable device for the clamping or sealing of the first structure4010 and the second structure 4012 together, etc. For example, thealignment holes 4089 of the second structure 4012 may be placed onto aclamping block (not shown), and the first structure 4010 may be foldedonto the second structure 4012. The clamping block or any other suitabledevice may then be used to clamp and seal the structures together. Thefirst structure 4010 and/or the second structure 4012 may be providedwith a structure tab such as a second structure tab 4012 a, which mayaid in the handling of the first and second structures, for example.

Boundary material (referred to herein as “boundary material” or“boundary spacers”) may be provided within first structure 4010 and/orthe second structure 4012. After the substrate is folded, such boundarymaterial may form at least a portion of a perimeter of the sealedchamber. It should be understood that the boundary material may extendalong a perimeter of the second structure 4012 such that the channel4022 may be sealed from all its sides. In some variations, the boundarymaterial may be provided within both the first structure 4010 and thesecond structure 4012, in any suitable pattern. In the exemplaryembodiment of the chamber arrangement shown in FIG. 40A, the firststructure 4010 is provided with a ring-shaped boundary material 4021 a,which may receive and encircle the boundary material 4021 b of thesecond structure 4012 when the device is in a folded state (FIG. 40B).The channel 4022 may thus be placed in the recess 4022 a of the firststructure 4010, within the boundary material 4021 a. In the exemplaryembodiment of the chamber arrangement shown in FIG. 40C, the firststructure 4010 may be provided with boundary material 4021 a throughoutthe structure, wherein the boundary material 4021 a extends throughoutthe cover with the exception of the recess 4022 a. The boundary material4021 b of the second structure 4012 and the channel 4022 may, as in theembodiment shown in FIG. 40A, be placed in the recess 4022 a when thedevice is in the closed state. FIG. 40C shows a cross-sectional view ofthe single piece fluid device of FIG. 40A in a folded state. When thesingle piece fluid device is in a fully folded state, the fluid channelmay be sealed. As shown in FIG. 40A, at least one inlet (“I”) and atleast one outlet (“0”) for the fluid chamber 4022 may provide fluidicaccess to the fluid chamber 4022 from one of the first and secondstructures, such as the second structure 4012 of the single piece fluiddevice, for example.

In other words, in some variations, at least a portion of boundarymaterial in the first structure and at least a portion of the boundarymaterial in the second structure may be complementarily formed so as tomate when the chamber arrangement is in the closed state, and form asealed chamber within the mated boundary material. Furthermore, itshould be understood that different portions and patterns of theboundary material may be provided in the first and second structure ofthe chamber arrangement, in addition to the exemplary patterns shown inFIGS. 40A and 40C.

In some variations, all of the boundary material may be provided in oneof the first and second structures. For example, as shown in the sidecross-sectional views of a chamber arrangement depicted in FIGS. 42A and42B (showing partially closed and closed states, respectively), theboundary material 4021 b may be provided solely within the secondstructure 4012, while the first structure 4010 may form a cover withoutboundary material deposited thereon. As shown in FIG. 42B, the firststructure 4010 may lie flush against the top of the boundary material4021 b (and spacers 4024) when the device is in the closed state. Again,as previously described, when the single piece fluid device is in afully closed state, the fluid chamber 4022 may be sealed.

Although specific exemplary variations of chamber arrangements aredescribed above with references to FIGS. 2A-8B and FIGS. 40A-40C, 41,and 42A-42B, it should be understood that various features of thesechamber arrangements may be combined in any suitable manner.

Electromerging Chamber

In some variations, the chamber may be configured to alter at least aportion of the sample in the chamber, such as by merging two or moreentities or particles in the sample through application of electricalenergy by electrodes (“electromerging”). Such electromerging may, asdescribed in further detail below, enable further processing such assorting and separating particles by size in order to efficiently isolatecertain particles of interest for further processing. For example,electromerging chamber arrangements may be used to identify and sortcells of interest, such as cells (e.g., hybridomas, B cells, Chinesehamster ovary (CHO) cells, etc.) that are high secretors of desiredsubstances such as specific antibodies or insulin for development ofimmunotherapy treatments, etc.

For example, as shown in FIG. 23A, a method 2300 of processing a samplemay include receiving a prepared emulsion including a plurality ofparticles in a chamber 2320, characterizing one or more particles in thesample as discard particles 2330, merging at least a portion of thediscard particles 2340 by delivering electrical energy to the discardparticles, and sorting particles based on particle 2350. Furtherprocessing 2360 may be performed on certain particles of interest, suchas collecting particles of interest into a reservoir, pipetting orotherwise depositing PODS or cells into a well plate, performingcellular PCR, DNA sequencing, ELISA, FACS, and/or other processing, etc.

FIG. 23B depicts an exemplary schematic of preparation of a sampleincluding particles (e.g., PODS) of interest among a plurality ofparticles. Specifically, in this example, the sample includes cellsmixed with a surfactant (e.g., fluoro-oil) and beads or other markers tocreate an emulsion with PODS, where each POD may function as aself-contained vesicle. For example, as shown in FIG. 31, fluoro-oil3120 may be added to an emulsion 3130, and the emulsion 3130 may beintroduced into an electromerging chamber 3110. Oil 3120 mayadditionally be introduced separately into the electromerging chamber3110. The sample may be polydisperse with PODS of different sizes. Acell may secrete one or more antibodies. For example, as shown in FIG.23B, cell C1 may secrete a first type of antibody (ab1), cell C2 maysecrete a second type of antibody (ab2), and cells C3 and C4 may secretea third type of antibody (ab3). These cells may be mixed with beadscoated with antigens specific to an antibody type of interest (e.g.,ab3) such that in the mixed sample, the beads bind to secretor cells ofinterest, and the resulting agglutination may be an indicator of thepresence of the cells of interest and/or of the level of secretion bythe cells of interest. In the resulting emulsion, each POD may includeat least one cell (which may or may not be a cell of interest) or lackcells. For example, PODS P1-P3 may include cells C1-C3, respectively,while other PODS (Pe) may lack cells. In some variations, the ratio ofcells to surfactant may be low so as to produce a relatively diluteconcentration of PODS with cells. For example, the average number ofcells per POD (λ) may be between about 0.9 and about 1.1, or about 0.1(e.g., about 10 “empty” PODS without cells for every 1 POD with at leastone cell). Furthermore, only a fraction of these cells may be cell ofinterest (that is, secreting an antibody of interest), and only afraction of those cells may be desirable cells of interest (that is,secreting the antibody of interest at a sufficiently high rate) suitablefor further processing such as for immunotherapy. Exemplary systems andmethods for extracting the desirable cells of interest from the emulsionare further described below with respect to FIGS. 23A-24B. FIG. 31illustrates another exemplary variation of the system.

One advantage of the electromerging chamber arrangement combined withPODS is that each POD serves as a low volume vesicle (e.g., betweenabout 500 picoliters-1 nanoliter in volume, on average) which enablesreadout and identification of a low amount of antibodies (or othersubstance of interest). In some variations, cells of interest mayrequire only up to a few hours to grow and secrete before they aresuitable for identification and sorting with the electromerging chamberarrangement. As an illustration, FIGS. 25A-25C are images depictinghybridoma growth (with IgG clustering) over time in a cell secretionassay, as captured by an imager and chamber systems such as thatdescribed herein. At time t=0, no agglutinates are visible (FIG. 25A).At this time, the concentration of IgG is low and below a secretionrange, as suggested by the visual similarities between FIG. 25A and FIG.26A (IgG concentration 0.5 ng/ml). However, after a mere two hours attime t=2 hours, hybridoma growth is already reflected by a sufficientlyhigh concentration of IgG within a hybridoma secretion range, assuggested by the visual similarities between FIG. 25B and FIG. 26B (IgGconcentration 5 μg/ml). A few more hours of growth at time t=6 hoursresults in IgG clustering that is even more easily detectable using theimaging and chamber systems described herein. Accordingly, theelectromerging chamber arrangement may provide a significantly fastermethod of producing antibodies or other substances of interest, incontrast to conventional production protocols which may require manydays (e.g., 10-14 days) of careful incubation during which cells must becloned and grown to sufficiently amplify the signal associated withagglutination.

Additionally, the sample may be introduced into the electromergingchamber arrangement as continuous flow, thereby enabling highthroughput. Furthermore, the output of the electromerging chamberarrangement has less dilution of particles of interest in the outputtedfluid volume. For example, in some variations, each well of a well platemay receive a single POD which is known to contain a high secretor celldrawn from the outputted fluid volume, compared to conventionalproduction protocols which typically result in many empty wells alongwith a few wells containing useful cells.

Generally, as shown in FIGS. 23C and 31, the sample may be introducedinto a chamber similar to chambers discussed above, except that thechamber includes one or more electrodes in the flow path of the sample.As described above with respect to FIG. 23A, processing in the chambermay be characterized as a multi-step process, including characterizing,merging, and sorting PODs. For characterizing PODS, an imager array mayobtain one or more images of the PODS in the sample, and the images maybe analyzed using computer vision and/or other computational techniquessuch as those described above, to characterize PODS based on theircontent (2330). For example, some PODS may be characterized as PODS ofinterest (e.g., containing cells that secrete antibodies of interestand/or that secrete the antibodies of interest at a sufficiently highrate) based on amount of agglutination present in the PODS. Other PODSmay be characterized as PODS for discard.

PODS characterized as discard PODS may then be merged to form largerPODS that are also intended for discard, by delivering electrical energyfrom one or more electrodes in contact with the discard PODS (2340).Electrodes and discard PODS may be capacitively coupled such thatvariations in voltage applied by the electrodes cause mechanicaldisturbances or other forces on the surfactant surfaces of PODS, therebybreaking the surface and causing adjacent PODS to rupture and mergetogether. For example, the electrodes in contact with the PODS may bedriven with an AC waveform such that the alternating modulation causes acyclical mechanical compression and decompression of the PODS surfactantsurfaces, thereby causing the PODS to rupture and merge. Suitable ACwaveforms include those described in further detail below with respectto FIG. 24B. Accordingly, the merging process (2340) may produce anemulsion in which PODS of interest are generally smaller, while PODSintended for discard are generally larger.

The electrodes in the chamber may have any suitable shape and/ororientation suitable for delivering electrical energy to particles inthe chamber. For example, the electrodes may be spacer posts 2402 (asshown in FIGS. 24-24C, for example) extending transverse to the sampleflow direction, such as extending between upper and lower surfaces ofthe chamber. As another example, the electrodes may be interdigitatedelectrodes 2810 as shown in FIGS. 28A and 28B, which are patterned on alower and/or upper surface of the chamber. In this example, FIG. 28Adepicts PODS prior to delivery of electrical energy via theinterdigitated electrodes 2810, and FIG. 28B depicts PODS including alarger, merged POD (P) that was created after delivery of electricalenergy via the interdigitated electrodes 2810.

Following such merging, the PODS in the emulsion may be sorted (2350)based on size in order to filter and isolate the PODS of interest whichare generally smaller. The sorting may be accomplished by any suitablesorting arrangement. In some variations, the sorting arrangement mayinclude a passive sorting arrangement. For example, the sortingarrangement may include multiple spacers (e.g., similar to spacer posts424 as in the chamber shown in FIG. 4A), which may be arranged in astaggered array and configured to perform separation via deterministiclateral displacement (DLD). In DLD, a staggered array of posts may worksimilar to a marble machine to separate small and large particles suchas PODS. For example, as shown in FIG. 23D, as a sample including smalland large particles generally move in a direction of fluid flow throughthe staggered array of posts, large particles (that is, particles over acritical threshold diameter) may be steered laterally relative to thefluid flow direction. Such passive migration of the larger particles maythus enable separate collection of smaller particles (e.g., at a firstoutlet) and larger particles (e.g., at a second outlet). The criticalthreshold diameter for sorting particles in this manner may be adjusted,for example, by tailoring the diameter and/or spacing of the posts.Accordingly, in some variations of a sorting arrangement, spacer postsin the chamber may be constructed and arranged so as to passively sortPODS in the sample by deterministic lateral displacement.

As another example, the sorting arrangement may include one or moreoutlets of various sizes that selectively permit passage ofdifferently-sized particles. For example, as shown in the schematic ofFIG. 23C, the chamber may include multiple small outlets (e.g., one ormore selection channels leading to capture at a “SelectPort”) configuredto only permit passage of particles below a predetermined thresholdparticle size, and reject passage of particles above the predeterminedthreshold particle size. One or more larger outlets (“TrashPort”)leading to a waste receptacle may be configured to permit passage oflarger particles that were rejected by the preceding smaller outlets.Thus, in a chamber such as that shown in FIG. 23C, smaller PODS may tendto exit the chamber through smaller outlets, before reaching the largeroutlet(s) for discard with the larger PODS. As another example, as shownin the schematic of FIG. 30, multiple channels leading to one or moreoutlets leading to a waste receptacle (“Trash”) may reject passage oflarger particles, which may be guided by channels toward a capturereceptacle (“Select”) that accepts passage of larger particles.Accordingly, in some variations of a sorting arrangement, an array ofprogressively increasing chamber outlet sizes may passively sort PODS inthe sample through collection according to particle size. As shown inFIG. 23C and FIG. 31, particles not of interest may be collected fordiscard (e.g., a waste receptacle at “TrashPort” shown in FIG. 23C, orwaste receptacle 3140 shown in FIG. 31). Furthermore, in somevariations, fluidic currents (e.g., constructed through use of pumps,valves, chamber surface contouring, etc.) may, additionally urgeparticles toward the smaller outlets (e.g., against sidewalls of themain channel) to further encourage sufficiently small particles to passthrough the smaller outlets.

As another example, as shown in FIG. 23E, the sorting arrangement mayadditionally include multiple branching channels configured to performparticle separation via hydrodynamic filtration. In hydrodynamicfiltration, a small amount of fluid is withdrawn repeatedly from themain channel through one or more side branching channels, whichgradually concentrates and aligns particles along the sidewalls of themain channel. The concentrated and aligned particles can then becollected according to particle size through one or more selectionchannels, similar to that described above and shown in FIG. 23C.

Additionally or alternatively, the sorting arrangement may include anactive sorting arrangement. For example, the chamber may include one ormore electrode regions configured to generate electrical fields toenable dielectrophoresis, such as similar to those described in U.S.patent application Ser. No. 15/986,416 which was incorporated byreference above. Such electrode regions may, for example, be operated tocapture, move, and/or otherwise actively control sorting of selectedPODS.

After sorting PODS and collecting PODS of interest, the PODS of interestmay be further processed. For example, the smaller PODS of interest maybe directed (e.g., via vacuum or other aspects of a fluidic controlsystem as described below) into a reservoir (2360), from whichindividual PODS or cells may be withdrawn (2363) with a pipette or otherinstrument. PODS of interest may be deposited into well plates forfurther processing and/or analysis (e.g., PCR, sequencing, etc.). Forexample, up to a single cell may be deposited in each well. Aprogrammable robot may automatically load each well, thereby furtherincreasing efficiency.

FIGS. 24A-24C are schematic illustrations of an exemplary variation ofan electromerging chamber arrangement. As shown in FIG. 24A, a systemfor processing a sample may include a chamber 2400 including at leastone inlet 2410 for receiving a sample (e.g., through tubing and asuitable fluidic connection), and two or more outlets (e.g., 2420 and2422) for allowing at least a portion of the sample to exit the chamber2400. Along the flow path between the inlet 2410 and the outlets 2420,2422, the chamber may include an imaging and merging region 2402, and asorting region 2404 that is downstream from the imaging and mergingregion 2402. The chamber 2400 may include multiple spacer posts 2432 and2434, distributed throughout regions 2402 and 2404 respectively, thatsupport and/or maintain the gap distance between upper and lowersurfaces of the chamber. For example, as shown in the cross-sectionalstack-up schematic FIG. 24B, the spacer posts 2432, 2434 may extendbetween and support the spacing (e.g., 75 μm or other suitable gapdistance) between transparent polyimide surface (having a thickness ofabout 25 μm, or other suitable thickness). The chamber may, in somevariations, be formed with a lamination press that joins the polyimidesurfaces at least in part with a suitable adhesive.

As described in further detail below, at least the spacer posts 2402 inthe imaging and merging region 2402 may function as electrodes thatdeliver electrical energy to merge selected PODS. The spacer posts 2432,2434 may, for example, include a conductive material such as copper. Atleast the spacer posts 2404 in the sorting region may function to sortPODS according to size.

Generally, the imaging and merging region 2402 may be positioned betweenone or more light sources and/or imager array such that the imager arraymay obtain shadow images of PODS or other particles that have enteredthe chamber. The one or more images may be analyzed using computationtechniques such as those described herein with respect to FIGS. 21A-21E.Based on the analysis of such images, PODS that are not of interest(e.g., do not include cells, or include cells lacking IgG or otheragglutination) may be characterized as PODS for discard. Such discardPODS may then be designated for electromerging by one or more processorsin the system.

Electromerging may be accomplished with spacer posts 2402 functioning aselectrodes. As shown in FIG. 24C, an electrode may be conductivelycoupled (e.g., through a conductive connection such as a trace orwiring) to a controller 2450 configured to control the activation of theelectrodes. Although FIG. 24C illustrates four conductive connectionsfor respectively four electrodes, it should be understood that in somevariations, more than four spacer posts 2402 may function as electrodes,and each electrode may have its own respective conductive connection (oralternatively, at least some number n electrodes may be controlled byfewer than n conductive connections through a suitable multiplexingscheme).

In this example, the controller 2450 may include a signal generator 2456configured to generate one or more suitable waveforms with which todrive the electrodes. In some variations, the signal generator 2456 maybe configured to drive the electrodes with an AC waveform (e.g., square,triangle, sinusoidal, etc.), such that PODS between pairs of electrodes(e.g., adjacent pairs) are capacitively coupled to the electrodes andreceive electrical energy with alternating polarity, Upon receiving suchelectrical energy, the PODS experience periodic compressive forces thatbreaks the PODS and causes adjacent affected PODS to merge into largerpod(s). Specific parameters of the AC waveform may vary depending on theapplication (e.g., size of PODS, size and material of electrodes,spacing between electrodes, etc.), but generally the drive waveformshould have sufficient voltage to elicit the merging effect, withoutbeing so excessive so as to result in damage to the sample (e.g., resultin bubbles, black spots, etc.). For example, in some variations thewaveform may have a peak-to-peak voltage between about 0.5 V and about10 V, between about 0.5 V and about 5 V, or about 2.5 V. Furthermore, insome variations the waveform may have a frequency between about 1 Hz and1 MHz, between about 10 Hz and about 20 kHz, or between about 50 Hz andabout 20 kHz. For a single instance of merging PODS, the pulses of thedrive waveform may cycle any suitable number times, such as betweenabout 1 and 20 times, and pulse width may, in some variations, varybetween about 10 ms and 10 s in duration. However, the drive waveformmay have any suitable pulse width, number of cycles, etc.

The signal generator 2456 may be conductively coupled to each electrodewith traces, wiring, or other suitable connection. Along theseconductive connections, signal processing circuitry 2454 (e.g.,amplifiers) may, for each individual conductive connection orcollectively for all conductive connections, may amplify or otherwisemodify the driving signal as appropriate. Furthermore, a switch array2452 including a switch for each conductive connection may be controlledto selectively turn ON and OFF the activation of each switch'scorresponding electrode. Accordingly, the controller 2450 may cause atleast some of the spacer posts 2402 (functioning as electrodes) todeliver suitable electrical energy for electromerging PODS that areidentified for merging (e.g., PODS not of interest) and are in contactwith or capacitively coupled with the spacer posts 2402.

As described above, following the passage of the sample through theimaging and merging region 2402 of the chamber, the larger PODS aregenerally PODS not of interest (e.g., do not include high secretorcells) while the smaller PODS are of interest and are desirable to keep.Therefore, the sorting region 2404 of the chamber functions to separatethe smaller PODS from the larger PODS for collection. As shown in thevariation depicted in FIGS. 24-24C, the sorting region 2404 may includespacer posts 2434 arranged in a staggered array, which may be configuredto passively sort larger PODS laterally (relative to the left-to-rightflow direction) toward outlet 2422. Smaller PODS may simultaneously bepassively sorted toward outlet 2420. It should be understood, however,that in other variations the electromerging chamber may additionally oralternatively include any suitable passive and/or active particlesorting arrangement.

Accordingly, by directing the sample across the imaging and mergingregion 2402 and the sorting region 2404, the electromerging chamberarrangement 2400 may provide a concentrated output of PODS of interest(collectable at outlet 2422), and a separate, waste output of PODS notof interest (collectable of outlet 2420) that avoids dilution of thePODS of interest. Furthermore, a continuous flow of sample through thechamber 2400 may enable a high throughput of PODS, thereby furthercontributing to a highly efficient processing of samples, suggestingviability of the electromerging systems and methods described herein.

Fluidic Control System

As shown in the schematic of FIG. 1A and the illustration showing anexemplary variation in FIG. 1B, the system 100 may include a fluidiccontrol system configured to manipulate PODS with a pressuredifferential. The fluidic pressure differential may induce one or morePODS to enter the chamber through the chamber inlet 122, include one ormore PODS to traverse across the chamber, and/or induce one or more PODSto exit the chamber through the chamber outlet 124. For example, thesystem 100 may include at least one positive pressure pump 110fluidically coupled to (or otherwise associated with) the chamber inlet122 and/or at least one negative pressure pump 150 fluidically coupledto (or otherwise associated with) the chamber outlet 124. The pumps 110and/or 150 may be configured to draw an emulsion (including PODS, forexample) from a reservoir 116 (e.g., tank, Eppendorf tube, othersuitable container, etc.) into the chamber 120 via tubing and the atleast one chamber inlet 122. The pumps 110 and/or 150 may additionallyor alternatively be configured to draw at least a portion of theemulsion from the chamber 120 through the at least one chamber outlet124. In some variations, a waste container 156 may be coupled in-linebetween the chamber outlet 124 and the pump 150 for receiving andholding emulsion that has exited the chamber 120. Although the schematicof FIG. 1 illustrates one pump 110 associated with one chamber inlet112, and one pump 150 associated with the chamber outlet 124, it shouldbe understood that in other variations, the system may include anysuitable number of chamber inlets, chamber outlets, and pumps.Furthermore, in some variations, the chamber 120 may be detachable forintegration with other fluidic control systems. The chamber 120 may be adisposable component, while the rest of the fluidic control system maybe reusable and/or sterilizable.

Additionally, the assay system 100 may include one or more valves thatmay enable further fluidic control within the assay system 100. Forexample, valve 112 may be located in-line with fluidic flow to one ormore chamber inlets, and may be controlled to regulate sample flow intothe chamber 120. Additionally or alternatively, valve 152 may be locatedin-line with fluidic flow from one or more chamber outlets, and may becontrolled to regulate sample flow out of the chamber 120. Furthermore,the assay system 100 may include one or more pressure sensors 114, 154(or flow sensors, or any suitable sensors) configured to monitorpressure and/or other parameters of the fluidic system.

In some variations, components of the fluidic control system, includingthe above-described pumps, valves, and/or sensors, can be controlled byone or more controllers. For example, the electronics system 160 mayinclude one or more controllers configured to implement any suitablecontrol system to operate one or more pumps and/or valves based at leastin part on sensor input from the pressure sensors, to maintain a desiredrate of flow into the chamber 120. Furthermore, the control system canoperate these components so as to facilitate sorting of the samples inthe chamber, as further described in U.S. patent application Ser. No.15/986,416 which was incorporated by reference above.

Electronics System

As shown in FIG. 1, the system 100 may include an electronics system160. The electronics system 160 may include, for example, a PCBA withone or more processors, etc. configured to control and/or receivesignals from other components of the assay system 100, as furtherdescribed herein. In some variations, the electronics system 160 mayfurther include one or more communication components (e.g., Bluetooth,WiFi, etc.) configured to communicate data (e.g., image data) to anetwork 170 for analysis by one or more remote processors 180. Forexample, the network 170 may include any suitable wired or wirelessconnection with one or more computing devices. Additionally oralternatively, at least some of the data may be analyzed by one or moreprocessors located in the electronics system 160.

Generally, the computing devices may include a controller including aprocessor (e.g., CPU) and memory (which can include one or morecomputer-readable storage mediums). The processor may incorporate datareceived from memory and user input. The memory may include storeinstructions to cause the processor to execute modules, processes,and/or functions associated with the methods described herein. In somevariations, the memory and processor may be implemented on a singlechip, while in other variations they can be implanted on separate chips.

The processor may be any suitable processing device configured to runand/or execute a set of instructions or code, and may include one ormore data processors, image processors, graphics processing units,physics processing units, digital signal processors, and/or centralprocessing units. The processor may be, for example, a general purposeprocessor, a Field Programmable Gate Array (FPGA), an ApplicationSpecific Integrated Circuit (ASIC), and/or the like. The processor maybe configured to run and/or execute application processes and/or othermodules, processes and/or functions associated with the system and/or anetwork associated therewith. The underlying device technologies may beprovided in a variety of component types (e.g., MOSFET technologies likecomplementary metal-oxide semiconductor (CMOS), bipolar technologieslike emitter-coupled logic (ECL), polymer technologies (e.g.,silicon-conjugated polymer and metal-conjugated polymer-metalstructures), mixed analog and digital, and/or the like.

One or more processors may, for example, provide a computer visionsystem configured to analyze images (e.g., shadow images acquired asdescribed herein) to assess an imaged sample using suitable imageprocessing and/or computer vision techniques. For example, withreference to FIGS. 21A-21E, in some variations, one or more processorsmay process an original (raw) shadow image (FIG. 21A) to reduce noise(e.g., through a filter process) and remove background content (FIG.21B) (e.g., obtained through a control image of the chamber when emptyor without a sample under the same or similar lighting conditions, orwith a software algorithm). The sample image may be subtracted from thebackground image to obtain a subtracted image (FIG. 21C). Aftersubtraction, any dark objects in the original image may appear brighterin the subtracted image. The subtracted image may be thresholded onpixel intensities to obtain a binary, black and white image of one ormore PODS (FIG. 21D). For example, any objects in the POD may appearwhite in the binary image, while other regions may appear black (orvice-versa). Finally, suitable computer vision techniques (e.g., contoursearching algorithm) may be applied to find the boundaries of object(e.g., POD, POD content such as cells or particles), thereby enablingidentification of the object. In some variations, such contour searchingalgorithms may incorporate one or more trained machine learning models.One or more characteristics of POD and/or POD content based on suitablecomputer vision techniques may be identified in the processed image(e.g., as highlighted in FIG. 21E). For example, many properties such asarea, particle size, particle shape, greyscale (e.g., intensity), rateof movement, current flow within the POD, ratios and/or dynamic changesthereof, and/or any combination thereof may be analyzed.

In some variations, the memory may include a database and may be, forexample, a random access memory (RAM), a memory buffer, a hard drive, anerasable programmable read-only memory (EPROM), an electrically erasableread-only memory (EEPROM), a read-only memory (ROM), Flash memory, andthe like. The memory may store instructions to cause the processor toexecute modules, processes, and/or functions such as measurement dataprocessing, measurement device control, communication, and/or devicesettings. Some variations described herein relate to a computer storageproduct with a non-transitory computer-readable medium (also may bereferred to as a non-transitory processor-readable medium) havinginstructions or computer code thereon for performing variouscomputer-implemented operations. The computer-readable medium (orprocessor-readable medium) is non-transitory in the sense that it doesnot include transitory propagating signals per se (e.g., a propagatingelectromagnetic wave carrying information on a transmission medium suchas space or a cable). The media and computer code (also may be referredto as code or algorithm) may be those designed and constructed for thespecific purpose or purposes.

Examples of non-transitory computer-readable media include, but are notlimited to, magnetic storage media such as hard disks, floppy disks, andmagnetic tape; optical storage media such as Compact Disc/Digital VideoDiscs (CD/DVDs); Compact Disc-Read Only Memories (CDROMs), andholographic devices; magneto-optical storage media such as opticaldisks; solid state storage devices such as a solid state drive (SSD) anda solid state hybrid drive (SSHD); carrier wave signal processingmodules; and hardware devices that are specially configured to store andexecute program code, such as Application-Specific Integrated Circuits(ASICs), Programmable Logic Devices (PLDs), Read-Only Memory (ROM), andRandom-Access Memory (RAM) devices. Other variations described hereinrelate to a computer program product, which may include, for example,the instructions and/or computer code disclosed herein.

The systems, devices, and/or methods described herein may be performedby software (executed on hardware), hardware, or a combination thereof.Hardware modules may include, for example, a general-purpose processor(or microprocessor or microcontroller), a field programmable gate array(FPGA), and/or an application specific integrated circuit (ASIC).Software modules (executed on hardware) may be expressed in a variety ofsoftware languages (e.g., computer code), including C, C++, Java®,Python, Ruby, Visual Basic®, and/or other object-oriented, procedural,or other programming language and development tools. Examples ofcomputer code include, but are not limited to, micro-code ormicro-instructions, machine instructions, such as produced by acompiler, code used to produce a web service, and files containinghigher-level instructions that are executed by a computer using aninterpreter. Additional examples of computer code include, but are notlimited to, control signals, encrypted code, and compressed code.

In some variations, a computing device may further include acommunication interface configured to permit a patient and/or other usedto control the computing device. The communication interface may includea network interface configured to connect the computing device toanother system (e.g., Internet, remote server, database) by wired orwireless connection. In some variations, the computing device may be incommunication with other devices via one or more wired or wirelessnetworks. In some variations, the communication interface may include aradiofrequency receiver, transmitter, and/or optical (e.g., infrared)receiver and transmitter configured to communicate with one or moredevice and/or networks.

Wireless communication may use any of a plurality of communicationstandards, protocols, and technologies, including but not limited to,Global System for Mobile Communications (GSM), Enhanced Data GSMEnvironment (EDGE), high-speed downlink packet access (HSDPA),high-speed uplink packet access (HSUPA), Evolution, Data-Only (EV-DO),HSPA, HSPA+, Dual-Cell HSPA (DC-HSPDA), long term evolution (LTE), nearfield communication (NFC), wideband code division multiple access(W-CDMA), code division multiple access (CDMA), time division multipleaccess (TDMA), Bluetooth, Wireless Fidelity (WiFi) (e.g., IEEE 802.11a,IEEE 802.11b, IEEE 802.11g, IEEE 802.11n, and the like), voice overInternet Protocol (VoIP), Wi-MAX, a protocol for e-mail (e.g., Internetmessage access protocol (IMAP) and/or post office protocol (POP)),instant messaging (e.g., extensible messaging and presence protocol(XMPP), Session Initiation Protocol for Instant Messaging and PresenceLeveraging Extensions (SIMPLE), Instant Messaging and Presence Service(IMPS)), and/or Short Message Service (SMS), or any other suitablecommunication protocol. In some variations, the devices herein maydirectly communicate with each other without transmitting data through anetwork (e.g., through NFC, Bluetooth, WiFi, RFID, and the like).

Clustering Assays

As discussed above, systems and methods such as those described hereinmay, for example, be used for processing cell samples. A clusteringassay may be used for identification of cells that are high secretors orproducers of antibodies for specific targets, for example. In apopulation of cells, certain cells within the population may, relativeto the other cells of the population, be high secretors of a target ofinterest, such as a protein of interest. Exemplary variations of systemsand methods for clustering assays are described in further detail below.

“One-Bead” Assay

A clustering assay using a one-bead system (“one-bead assay,” or“one-bead clustering assay”) may be used to identify the high secretorcells within the population of cells. Although the “one-bead assay” isprimarily described below as including one or more beads as markerparticles, it should be understood that other particles may be used(e.g., cells, as described in further detail below). The one-beadclustering assay may be used to identify a secreting cell encapsulatedwithin a POD as a high secretor of a target protein (or peptide, etc.).For example, the assay may utilize one or more particles of a first typethat provides a signal indicating a cell's secretion level of theantibody of interest.

FIG. 32A depicts a schematic diagram of the binding interactions thatmay occur inside of a sample entity such as a POD 3218 when using aone-bead assay. The POD 328 may include one or more particles with abinding partner that may be specific to a second binding partner that issecreted by a cell 3217. FIG. 32B depicts a detailed enlargement ofregion 3220 of FIG. 32A, showing a single particle 3211 a. As shown asan example, the one-bead clustering assay may be used to identify anantibody secreting cell 3217 encapsulated within a POD 3218 as a highsecretor of the antibody of interest 3213. The one-bead clustering assaymay utilize an encapsulation reagent, and a first plurality of particlessuspended in aqueous media. The encapsulation reagent may comprise adensity greater than about 1.0 which may, for example, help formdiscrete sample entities such as PODs including aqueous media. Eachparticle of the first plurality of particles may comprise a firstbinding partner that is specific to a second binding partner secreted bythe cell of interest.

Again, as was previously described when referring to FIG. 23B, a samplefor use in a one-bead assay may include cells mixed with a surfactantsuch as a fluoro-oil and beads or other markers to create an emulsionwith sample entities, where each sample entity may function as aself-contained vesicle. The encapsulation reagent may include thesurfactant, which may at least in part enable the encapsulation ofportions of the sample into the sample entities. Examples offormulations wherein the encapsulation reagent comprises a surfactantare shown in Tables 1-3. The PODS may be polydisperse within a sampleused for analysis. The particles of the first plurality of particles maybe beads, such as an antibody coupled bead 3211 a as shown as an examplein FIG. 32B, having a polyclonal antibody 3212 a coupled to its surface.The particles of the first plurality of particles may be cells, suchthat antigens or any other suitable markers expressed on the cell maybind with the antibody secreted by a cell of interest. The markers orantigens on a cell or the polyclonal antibody 3212 a on a bead may thusact as the first binding partner that is specific to a second bindingpartner, which may be a binding domain or any suitable component 3213 aof the antibody 3213 secreted by the cell 3217. In some variations, thefirst binding partner may comprise a first protein, and the secondbinding partner may comprise a second protein. In some variations, thefirst binding partner or the second binding partner may be an antigen orantibody. In some variations, the first binding partner may comprise afirst peptide, and the second binding partner may comprise a secondpeptide.

When the first binding partner 3212 a and the second binding partner3213 a are bound, the site of the binding may be referred to as acluster site, or a first cluster site 3214 a as shown in FIG. 32B. Thesecluster sites formed by a binding of the first and second bindingpartners may be detected by the systems and methods such as thosedescribed herein. Furthermore, in some variations, certain cells may beselected as being a cell of interest by being a high secretor of theantibody 3213, and high secretor cells may generate larger clusters. Theclusters formed by the first binding partner 3212 a and the secondbinding partner 3213 a may be observable using a lensless imager systemas described above, for example, and thus may enable the selection of ahigh secretor cell. High secretor cells may also be measured or detectedusing any of the imaging methods described herein.

Thus, the clustering assay may enable identification of PODs includingcells of interest (e.g., cells that secrete a sufficiently high amountof a substance of interest). For example, a larger amount of theantibody of interest being secreted from a cell may lead to a largercluster due to a larger amount of cluster sites being formed, such asthe first cluster sites shown by 3213 a of FIG. 32B. A larger amount ofinteractions between the polyclonal antibody 3212 a coupled to the bead3211 a and the binding region 3213 a of the antibody 3213 secreted bythe cell may thus result in a larger number of clusters being formed,which may then be detectable as one or more large clusters. The formedclusters may then be visually detected, or measurable by any of theimaging methods described herein, and may be interpreted as a signalthat the POD from which the cluster is detected is a POD containing acell of interest.

Generally, a larger cluster will result in a POD having ahigher-secreting cell of the antibody of interest. The larger size ofthese clusters may allow for the clusters to be visualized using a 4×microscope objective, for example. As another example, the clusters maybe observed in shadow images such as those taken by the systemsdescribed above with reference to FIGS. 2A, 9, 10, and 14A-14C. Anexample of a cell secreting or producing a sufficient amount of anantibody of interest to generate a detectable or measurable signal isshown in a first POD 3218 a of FIG. 32A.

In some variations, a POD comprising one or more cluster sites may beassigned a particle size score (PSS). For example, PSS may be determinedfor and assigned to a POD as described below in the Examples. PODs withhigher PSS, for example, may be identified as including a cell ofinterest (e.g., high secretor cell) and may be sorted (e.g., asdescribed above through electromerging and sorting processes, etc.) soas to separate cells of interest from the population of cells. At leastsome of the sorted cells may undergo further processing, such as ELISA,FACS, DNA sequencing, PCR, other suitable analysis, and so on. In somevariations, the cell of interest may be removed from the POD 3218 forsuch further processing.

The clustering assay also enables identification of PODs not includingcells of interest (e.g., cells that secrete no or low amounts of asubstance of interest). For example, when a cell within a POD is notsecreting the antibody of interest, no clusters may be formed due to nointeractions occurring between the polyclonal antibody 3212 a coupled tothe bead 3211 a and the antibody of interest. Additionally, when a cellwithin a POD is secreting a low amount of the antibody of interest,interactions may occur between the secreted antibody and the polyclonalantibody 3212 a coupled to the bead 3211 a, but the amount or number ofbinding interactions may be too low to generate a detectable ormeasurable signal. In some variations, a “low” signal may indicate lessthan about 1 pg of secreted amount of the antibody of interest overabout 3 hours, for a sample including PODS having an average volume ofabout 0.5 nL. For example, any formed clusters in a POD with alow-secreting cell may be below a threshold size, and/or may be toosmall to be visualized using a 4× microscope objective, or in shadowimages such as those described above with reference to FIGS. 2A, 9, 10,and 14A-14C. An example of a cell secreting or producing an insufficientamount of an antibody of interest to generate a detectable or measurablesignal is shown in a second POD 3218 b of FIG. 32A.

Examples of binding partners that may act as a first binding partner, asshown by 3212 a in FIG. 32B, may include a polyclonal antibody among oneor more classes of antibodies such as IgA, IgD, IgE, IgG, and IgM.

Examples of cells of interest that may be selected for using theone-bead assay may include any one or more of CHO cells, B cells,hybridoma cells, plasma cells, HEK293 cells, myeloma cells, and T-cells,etc.

Examples of antibodies of interest that may be selected for using theone-bead assay may include an antibody among one or more classes ofantibodies such as IgA, IgD, IgE, IgE, and IgM, etc.

Examples of proteins and peptides of interest includes any suitablestandard biomarkers such as insulin, NT-pro-BNP, Pro-GRP, β-CTX, PINP,pancreatic polypeptide, osteocalcin, β₂-microglobulin, calcitonin,cystatin C, C-peptide, VIP, ANF, NTX, β-amyloid (1-42), PSA, K-RAS,CA125, CA 15-3, MUC-1, HER-2/neu, estrogen receptor, progesteronereceptor, etc.

“Two-Bead” Assay

In some variations, it may also be valuable to identify and collectcells that are not only high secretors of a target of interest (again,such as a protein of interest) but also wherein the target of interestshows a high affinity and/or specificity of binding to a partnerantigen. A clustering assay using a two-bead system (“two-bead assay,”or “two-bead clustering assay”) may be used to assess the level ofantibody secretion from a target cell as well as the antigen bindingaffinity of the secreted antibody. Thus, the two-bead assay may be usedto select a cell of interest secreting a high amount of an antibody ofinterest, wherein the secreted antibody also displays a high antigenbinding affinity. For example, the assay may utilize one or moreparticles of a first type that provides a signal indicating a cell'ssecretion level of the antibody of interest, and one or more particlesof a second type that provides a signal indicating level of antigenbinding affinity for any such secreted antibody. Although the “two-beadassay” is primarily described below as including one or more beads asmarker particles, it should be understood that other particles may beused (e.g., cells, as described in further detail below).

Similar to the one-bead assay described above, the two-bead assay may beused for analysis of a sample, which may include cells mixed with asurfactant such as a fluoro-oil and beads or other markers to create anemulsion with sample entities, where each sample entity may function asa self-contained vesicle. The two-bead assay may also utilize anencapsulation reagent, and a first plurality of particles suspended inaqueous media, and a second plurality of particles also suspended inaqueous media. The encapsulation reagent may comprise a density greaterthan about 1.0. The encapsulation reagent may include a surfactant,which may at least in part enable the encapsulation of portions of thesample into the sample entities. The sample entities may be PODS.

Like with the one-bead assay described above, each particle of the firstplurality of particles may comprise a first binding partner that isspecific to a second binding partner secreted by the cell of interest.However, the two-bead assay may also utilize a second plurality ofparticles, wherein each particle of the second plurality of particlescomprises a third binding partner that is specific to a fourth bindingpartner secreted by the cell of interest.

FIG. 33A depicts a schematic diagram of the binding interactions thatmay occur inside of a POD 3318 when using a two-bead assay. Shown asexamples are PODS 3318 a, 3318 b, and 3318 d, as will be described infurther detail herein. POD 3318 a is an example of a detectable ormeasurable signal being generated, due to a cell 3317 identified as acell of interest, being a high secretor of an antibody of interest 3313,wherein the antibody 3313 has a high antigen binding affinity. POD 3318b is an example of no detectable or measurable signal being generated,due to a cell 3317 being a low producer of the antibody of interest. POD3318 d is an example of no detectable or measurable signal beinggenerated, due to a cell 3317 being a high secretor of the antibody ofinterest, but wherein the antibody 3313 has a low antigen bindingaffinity.

FIG. 33B depicts a detailed enlargement of POD 3318 a of FIG. 33A, andFIG. 33C shows a detailed enlargement of a binding interaction withinthe POD 3318 a of FIGS. 33A-33B.

In the two-bead assay, the particles of the first plurality of particlesmay be beads, such as an antigen coupled bead 3311 b having an antigen3312 b coupled to its surface. The antigen 3312 b may thus act as thefirst binding partner that is specific to a second binding partner,which may be a binding domain or any suitable first component 3313 a ofthe antibody 3313 secreted by the cell 3317. The first component 3313 amay be an antigen binding domain. The particles of the second pluralityof particles may also be beads, such as an antibody coupled bead 3315 bhaving an antibody coupled to its surface. The antibody 3316 may be amonoclonal antibody, and may act as the third binding partner that isspecific to a fourth binding partner, which may be a binding domain orany suitable component 3313 b of the antibody 3313 secreted by the cell3317. As shown in detail in FIG. 33C, when the antibody 3313 displays ahigh antigen binding affinity for the antigen 3312 b, coupled to thebeads making up the first plurality of particles 3311 b, a bindinginteraction may occur. When the antigen (acting as the first bindingpartner) 3312 b and a binding domain of the antibody (acting as thesecond binding partner) 3313 a are bound, the site of the binding may bereferred to as a cluster site, or a first cluster site 3314 a. A highamount of the antibody of interest 3313 being secreted by the cell 3317may cause a larger size of cluster formed by the interactions betweenthe antibody 3313 and the antigen 3312 b.

Additionally, a second binding domain 3313 b of the antibody 3313 maybind to a monoclonal antibody 3316 coupled to the beads making up thesecond plurality of particles 3315 b. A second cluster site 3314 b maybe formed by the binding of the binding domain of the antibody (actingas the third binding partner) 3316 and the monoclonal antibody (actingas the fourth binding partner) 3313 b.

Accordingly, the two-bead clustering assay may enable identification ofPODs including cells of interest (e.g., cells that are high secretors ofa substance of interest that also has a high specific level of bindingaffinity for another substance of interest). A POD having a cell thatsecretes a high level of the antibody of interest and wherein thesecreted antibody of interest has a high antigen binding affinity mayresult in a large cluster. A large cluster may be formed when both thefirst cluster site and the second cluster site are present throughout aPOD, such as the POD 3318 a shown as an example in FIG. 33A.

The two-bead clustering assay also may enable identification of PODs notincluding cells of interest (e.g., cells that secrete no or low amountsof a substance of interest and/or cells that secrete a substance ofinterest but the secreted substance of interest has a low bindingaffinity for another substance of interest). A POD having a cell thatdoes not secrete the antibody of interest, or a cell that secretes a lowamount of the antibody of interest, may result in a cluster below athreshold size and/or no measurable or detectable clustering, such asPOD 3318 b shown in FIG. 33A. Furthermore, a POD containing a cell thatis a high secretor of an antibody of interest, but wherein the antibodyof interest has a low antigen binding affinity, also may result in acluster below a threshold size and/or no measurable or detectableclustering, such as POD 3318 d shown in FIG. 33A. When using thetwo-bead assay to analyze a sample, the clusters formed by the bindingof the antibody to the monoclonal antibody coupled beads alone mayresult in clusters that are below a threshold size (e.g., measured by aparticle size score such as that described herein) or too small to bedetectable. The two-bead assay allows for an amplified signal caused bygrouping of several binding interactions together, such as in theexample POD 3318 a, wherein the antibody of interest is able to groupseveral beads together by its high affinity to the antigen 3312 b. Thus,the two-bead assay may be a useful tool for detection of a cell that isa high-secretor of an antibody of interest that has a high antigenbinding affinity and/or specificity.

In some variations, the first binding partner may comprise a firstprotein, and the second binding partner may comprise a second protein.In some variations, the first binding partner or the second bindingpartner may be an antigen or antibody. In some variations, the firstbinding partner may comprise a first peptide, and the second bindingpartner may comprise a second peptide. Furthermore, as described infurther detail below, in some variations the particles of the firstplurality of particles and/or second plurality of particles may becells, such as cells comprising one or more antigens or antibodies.

Certain cells may be selected as being a cell of interest by being ahigh secretor of the antibody 3313, wherein the antibody 3313 has a highantigen binding affinity and/or specificity, and thus may be selected bythe generation of a signal that is detectable using the imaging methodsdescribed herein. The detection or measuring of the signal may beperformed by specific detection of the monoclonal antibody 3316. Thecluster sites may also be detected or measured by the computer visionsystems and methods described herein.

In some variations, a POD comprising one or more cluster sites may beassigned a particle size score (PSS). For example, PSS may be determinedfor and assigned to a POD as described below in the Examples. PODs withhigher PSS, for example, may be identified as including a cell ofinterest (e.g., high secretor cell) and may be sorted (e.g., asdescribed above through electromerging and sorting processes, etc.) soas to separate cells of interest from the population of cells. At leastsome of the sorted cells, and/or their contents therein may undergofurther processing, such as ELISA, FACS, DNA sequencing, PCR, othersuitable analysis, and so on. In some variations, the cell of interestmay be removed from the POD 3318 for such further processing.

Examples of binding partners that may act as a first binding partner, asshown by 3312 b in FIG. 33C, may include an antigen.

Examples of cells of interest that may be selected for using thetwo-bead assay may include Chinese hamster ovary (CHO) cells, B cells,hybridoma cells, plasma cells, HEK293 cells, myeloma cells, and T cells,etc.

Examples of antibodies of interest that may be selected for using thetwo-bead assay may include an antibody among one or more classes ofantibodies such as IgA, IgD, IgE, IgE, and IgM, etc.

Examples of proteins and peptides of interest that may be selected forusing the two-bead assay may include insulin, NT-pro-BNP, Pro-GRP,β-CTX, PINP, pancreatic polypeptide, osteocalcin, β₂-microglobulin,calcitonin, cystatin C, C-peptide, VIP, ANF, NTX, β-amyloid (1-42), PSA,K-RAS, CA125, CA 15-3, MUC-1, HER-2/neu, estrogen receptor, progesteronereceptor, etc.

Preparation of Samples for Clustering Assay

FIGS. 34A and 34B depict exemplary variations of methods of preparing asample for a clustering assay. For example, FIG. 34A depicts a flowchart showing an exemplary method of preparing a sample for a one-beadclustering assay system. The method may include preparing a firstplurality of particles for use in the one-bead assay (3428 a), which mayinclude coupling the beads with polyclonal antibodies, and normalizingthe beads to a desired working concentration. The method may alsoinclude providing a population of cells, which may include at least onecell of interest (3421). The population of cells may be prepared bywashing in media and diluting to a desired cell concentration to createa cell dilution. Next, the method may include combining the populationof cells, the first plurality of particles, and an encapsulation reagentto create a mixture (3422), such as with a vortexer, stirrer (e.g.,magnetic stirrer), repeated pipetting, agitation, etc. Each particle ofthe first plurality of particles may comprise a first binding partnerthat is specific to a second binding partner secreted by the at leastone cell of interest, as further described above. The method may includeagitating the mixture to create an emulsion, thereby encapsulating thepopulation of cells into sample entities (3423). The sample entities maybe polydisperse (e.g., PODS, such as the POD 3218 c shown as an examplein FIG. 32B). The method may include incubating the emulsion (3427).Incubating the emulsion may, for example, allow sufficient time forcells of interest to secrete the substance of interest, if any, that mayinteract with the marker particles (e.g., first plurality of particles).Once incubated, the cells, encapsulated into the sample entities, may befurther analyzed such as by introducing the sample entities into theprocessing chamber as described in detail above, for visualization,assessment, merging, and/or sorting, etc.

FIG. 34B depicts a flow chart showing an exemplary method of preparing asample for a two-bead clustering assay system. The method may includepreparing a first plurality of particles and a second plurality ofparticles for use in the two-bead assay (3428 b), which may includecoupling the first plurality of beads with an antigen, and coupling thesecond plurality of beads with an antibody, and normalizing both batchesof beads to a desired working concentration. In some variations, thebead concentration is adjusted so that clustering is observedtypically >1 ng/mL or up to 100 ug/mL produced in 1 hour in a sampleincluding PODS having an average volume of 0.5 nL. The method mayinclude providing a population of cells, which may include at least onecell of interest (3421). The population of cells may be prepared bywashing in media and diluting to a desired cell concentration to createa cell dilution. The method may include combining the population ofcells, the first plurality of particles, the second plurality ofparticles, and an encapsulation reagent to create a mixture (3422 b).Similar to that described above, mixing may be performed with a vortexeror stirrer (e.g., magnetic stirrer, repeated pipetting, etc.). Eachparticle of the first plurality of particles may comprise a firstbinding partner that is specific to a second binding partner secreted bythe at least one cell of interest, and each particle of the secondplurality of particles may comprise a third binding partner that isspecific to a fourth binding partner secreted by the at least one cellof interest. The method may include agitating the mixture to create anemulsion, thereby encapsulating the population of cells intopolydisperse sample entities (3423). Again, the polydisperse sampleentities may be PODS. The agitating step (3423) may result in PODS suchas the POD 3318 c shown as an example in FIG. 33A. The method mayinclude incubating the emulsion (3427), similar to that described abovewith respect to the one-bead assay. Once incubated, the cells,encapsulated into the sample entities, may be further analyzed such asby introducing the sample entities into the processing chamber asdescribed in detail above, for visualization, assessment, merging,and/or sorting, etc.

In some variations, the encapsulation reagent for use in preparation ofthe sample for the one-bead and two-bead assays may comprise asurfactant. In some variations, the surfactant may comprise at least oneof fluorine and polyethylene glycol (PEG). In some variations, theencapsulation reagent may be between about 60% and 90% of the mixture.In some variations, the mixture may comprise one or more first particlessuspended in aqueous media, each first particle comprising a firstbinding partner. In some variations, the one or more first particles maybe between about 5% and 20% of the mixture by volume. In somevariations, the population of cells may be between about 5% and 20% ofthe mixture by volume. In some variations, the sample entities maycomprise polydisperse sample entities. In some variations, thepolydisperse sample entities may be PODS. In some variations, the firstbinding partner may comprise a first protein, and the second bindingpartner may comprise a second protein. In some variations, the firstbinding partner or the second binding partner may be an antigen orantibody. In some variations, the first binding partner may comprise afirst peptide, and the second binding partner may comprise a secondpeptide. In some variations, the population of cells may be CHO cells, Bcells, hybridoma cells, plasma cells, HEK293 cells, myeloma cells, or Tcells.

Table 1 shows an exemplary formulation of an emulsion sample that may beused in the method according to FIG. 34, or in any of the systems ormethods described herein, using a 1.5 ml Eppendorf to perform ananalysis run. Table 2 shows an exemplary formulation of a 250 ml sampleof PODS that may be used in the method according to FIGS. 34-35, or inany of the systems of methods described herein. Tables 1 and 2 do notaccount for any carrier fluid that may be present in the sample. Table 3shows an exemplary formulation for a complete sample test run, which maybe used in any of the systems and methods described here. In somevariations, the formulation summarized in Table 3 may be used forperforming an assay using a darkening substrate, as described herein.

TABLE 1 Exemplary emulsion sample formulation 1.5 ml Eppendorf % by % byVolume run Volume Weight Range Encapsulation % Fluoro-   75 (180 μl)82.0 60-180 μl reagents surfactant Detection % Bead 12.5 (30 μl) 9.010-30 μl reagents Sample media % water 12.5 (30 μl) 9.0 10-30 μl (cell)

TABLE 2 Exemplary 250 ml PODS sample % in 1 L % by % by Volume runVolume Weight Range Encapsulation % Fluoro-  75 (750 ml) 82.0 250 ml-750ml  reagents surfactant Detection % Bead 12.5 (125 ml) 9.0 41.67-125 mlreagents Sample media % water 12.5 (125 ml) 9.0 41.67-125 ml (cell)

TABLE 3 Exemplary sample formulation 1.5 ml Eppendorf % by % by Volumerun Volume Weight Range Encapsulation % Fluoro- 12.68 (180 μl) 12.88180-360 μl reagents surfactant Detection % Bead 2.11 (30 μl) 1.34 30-60μl reagents Sample media % water 2.11 (30 μl) 1.34 30-60 μl (cell)Carrier % carrier 70.42 (1 ml)  71.56 800-1000 μl Carrier % carrier,12.68 (180 μl) 12.88 180 μl post run rinse

As described hereinbefore, the particles of the first or secondplurality of particles used in the one-bead or two-bead assays may bebeads. Such beads may be polystyrene, gold, cellulose, latex, agarose,polyethylene glycol (PEG), glass, or magnetic beads. The beads may besuspended in aqueous media before and while being combined with thepopulation of cells in step 3422. The beads that act as the first andsecond plurality of particles may be polystyrene, gold, cellulose,latex, agarose, polyethylene glycol (PEG), glass, or magnetic beads, andmay be 10 nm to about 50 μm in size.

In some variations, a bead may comprise carboxylate, and may have adiameter between about 0.3 μm to about 6 μm, between about 0.05 μm andabout 20 μm, or between about 0.1 μm and 0.3 μm. In some variations, abead may comprise europium carboxylate, and may have a diameter betweenabout 0.10 μm to about 0.30 μm. In some variations, a bead may comprisecarboxyl-polystyrene, and may have a diameter between about 0.05 μm toabout 8 μm, or between about 1 to about 1.4 μm. In some variations, abead may comprise carboxylic acid groups, and may have a diameterbetween about 0.2 μm to about 5 μm, or may have a diameter of about 0.85μm, or about 0.4 μm.

In some variations, a cell may act as a particle of the first and secondplurality of particles. A cell may naturally express antigens, proteins,or other such markers on its cell surface, and these cell surfacemarkers may act as a first binding partner as in the interactionsdepicted in FIGS. 32B and 33C, or as a third binding partner as in theinteractions depicted in FIG. 33C. A cell expressing a marker on itssurface may therefore take the place of a bead in the one-bead ortwo-bead assays described herein.

In some variations, proteins having known binding partners or knowninteractions with other proteins may be utilized in the one-bead ortwo-bead assays. In these variations, an antibody may act as the firstbinding partner, such as in the exemplary embodiment shown in FIG. 32B,and may be coupled to a bead 4311 a by use of a bond (indicated at 4343)between known binding partners. Similar to the embodiment shown in FIG.32B, an antibody 4312 a, may be bound by a protein of interest secretedby a cell, for example, which may generate a measurable or detectablesignal for the assay. Again, this may indicate that the cell secretingthe protein of interest may be a high-secreting cell, and thus a cell ofinterest. In these variations, the antibody acting as the first bindingpartner 4312 a may be conjugated to a protein having a known bindingpartner. As shown in FIG. 43A as an example, the bead 4311 a may becoupled to biotin 4344. Streptavidin 4345, a known binding partner forbiotin 4344, may be conjugated to the antibody acting as the firstbinding partner 4312 a. The biotinylated bead may be provided separatelyfrom the antibody acting as the first binding partner. For example, aplurality of biotinylated beads may be provided as part of a kit, wherethe biotinylated beads may be mixed with streptavidin conjugatedantibodies acting as a first binding partner. Alternatively, thebiotinylated bead and the antibody may be provided together, such thatthe first binding partner is provided in a complex, including the bead4311 a, the biotin 4344, the streptavidin 4345, and the antibody 4312 a,wherein the biotin 4344 and the streptavidin 4345 are bound (indicatedat arrow 4343). As shown in FIG. 43B as another example, thestreptavidin 4345 may be coupled to the bead 4311 a, and the biotin 4344may be conjugated to the antibody 4312 a. Similar to the example shownin FIG. 43A, a plurality of beads 4311 a with streptavidin may beprovided as part of a kit, wherein the beads 4311 a may be mixed withbiotin conjugated antibodies acting as a first binding partner.Alternatively, the beads 4311 a coupled to streptavidin, and theantibody may be provided together, such that the first binding partneris provided in a complex including the bead 4311 a, the streptavidin4345, the biotin 4344, and the antibody 4312 a, wherein the streptavidinand the biotin are bound (indicated at 4343). In these examples, theplurality of beads may be used in a one-bead or two-bead assay, whereineach bead 4311 a of the plurality of beads is associated with a proteinsuch as biotin or streptavidin, and wherein the protein such as biotinor streptavidin is bound to a known binding partner (such as theexamples shown in FIGS. 43A-43B), and thus associated with the antibodyacting as the first binding partner 4312 a for the one-bead or two-beadassay.

In some variations, the antibody of interest secreted by a cell may bean IgG or other immunoglobulin (e.g., IgA, IgD, IgE, IgM, etc.).

In some variations, the reagents used in the one-bead and two-beadassays may include IVIES sodium salt, Tris, NaCl, Tween-20, and BSA, andvarious combinations thereof.

Exemplary Sample Preparations

An exemplary method of preparing a sample for use in a one-beadclustering assay system may be carried out as follows. As an example,and as previously described, the first plurality of particles maycomprise beads. Preparation of the beads as the first plurality ofparticles may include coupling the beads with an antibody andnormalizing the bead concentration. First, the process may includealiquoting the beads into a low-bind tube, pelleting the beads andremoving the supernatant. Next, the beads may be washed with a buffer,such as MES hemisodium salt, and the beads may next be resuspended infresh buffer. Several bead resuspensions may be prepared in this manner.EDAC (a water-soluble carbodiimide derivative) may be used at roomtemperature to make a 20× IVIES buffer with EDAC solution. The resultingEDAC solution may be added to each bead resuspension, which may then begently mixed and incubated at room temperature with rotation. Next, thebeads may be pelleted by centrifugation, washed with MES buffer,resuspended in fresh buffer and the antibody, and incubated. Afterincubation for approximately one and a half hours, blocking buffer maybe added. Next, repeated washing and incubation steps may be carried outto complete the antibody-coupling process. Finally, the beads may bewashed and resuspended in a storage buffer, at which stage theantibody-coupled beads may be stored at 4° C. for future use.

Next, to obtain beads having an appropriate, normalized concentrationfor use in the one-bead clustering assay, the beads may be normalized bymeasuring their absorbance as a representation of the concentration. Forexample, Nanodrop's OD600 function may be used to obtain the absorbance.A concentration standard such as a commercially available mouse IgGbeads standard, may be used for obtaining the beads concentration. Thebeads may be mixed by vortexing, measured using the Nanodrop forexample, and spun down, and next diluted or concentrated to a desiredconcentration.

The step of providing a population of cells, including at least one cellof interest (3421), may include preparation of a cell sample andcreating a cell dilution. This process may include counting the cellssuspension in the sample and checking for cell viability, and nextwashing the cells with ice cold media twice and resuspending the cellswith fresh ice cold media to a working concentration. The workingconcentration may be, for example, 4.4×10⁶ cells per ml. A working cellconcentration may then be made from this final cell concentration. Forexample, a 20-fold dilution may be performed to obtain the celldilution, having a working cell concentration of 2.2×10⁵ cells per ml.

Combining the population of cells, the first plurality of particles andthe encapsulation reagent may be performed by first mixing together thepopulation of cells (which may be provided as a cell dilution, preparedas described above) and the first plurality of particles (which may beprovided as antibody-coupled beads, prepared as described as above), forexample. A volume of the beads suspended in aqueous media such as abuffer as described above may be mixed with an equal volume of the celldilution. For example 30 μl of beads coupled with the polyclonalantibody (such as IgG) may be combined with 30 μl of the cell dilution.The mixing of the cell dilution and the beads may be performed byrepeated gentle pipetting, with a stirrer (e.g., magnetic stirrer), orthe like.

Next, the encapsulation reagent may be added to the mixed cell dilutionand antibody-coupled beads, to obtain the mixture for use in creating anemulsion. The encapsulation reagent may include a surfactant, which maybe a fluorosurfactant, for example. Examples of components of thesurfactant may include fluorine and polyethylene glycol (PEG), andfurther exemplary formulations are presented in Tables 1-3 below. Theemulsion may be obtained by agitating the mixture, which may beperformed by vortexing. The resulting emulsion may comprise sampleentities within which the cells and beads are contained. The sampleentities may be polydisperse sample entities, such as PODS, as describedherein. Thus, the agitating step (3423) may result in PODS such as thePOD 3218 c shown as an example in FIG. 32A.

The emulsion may be incubated in a fresh tube (for example, a 15 mlconical tube) with the encapsulation reagent, loosely capped in a cellincubator at 37° C. with 5% CO₂ for a predetermined length of time. Thepredetermined length of time may be between about 1 hour to about 6hours. After incubation, the emulsion may then be analyzed, such that acell of interest can be selected from the population of cells. Forexample, the emulsion may be loaded into a chamber as described hereinto read and analyze the PODS. The emulsion may also be visually observedusing, for example, a suitable objective system or the lenless imagersystem as described above.

An exemplary method of preparing a sample for use in a two-beadclustering assay may be similar to that described above with respect tothe method of preparing a sample for a one-bead clustering assay system,shown in FIG. 34A, except as described below.

The first plurality of particles and the second plurality of particlesmay comprise batches of beads. The first plurality of particles may beincubated with the antigen, and the second plurality of particles may beincubated with monoclonal antibodies, as in the method described above.Each batch of beads for the two-bead assay may next be normalized to adesired concentration as described, and a cell dilution may also beprepared as previously described. As an example, when creating themixture, about 15 μl of the antibody-coupled beads may be mixed withabout 15 μl of the antigen-coupled beads, to obtain a 30 μl bead volume.The 30 μl of beads, comprising the first and second pluralities ofparticles, may then be combined with an equal volume of the celldilution.

Performing a Clustering Assay

FIGS. 35A and 35B depict exemplary variations of methods of performing aclustering assay. FIG. 35A depicts an exemplary method of selecting atleast one cell of interest from a population of cells for use in aone-bead assay. As an example, at least one cell of interest may beselected from a population of cells using the following exemplarymethod. The method may include providing an emulsion comprising apopulation of cells and a first plurality of particles (3524), which maybe an emulsion prepared according to the method described whenreferencing FIG. 34A. Next, the method may include measuring a signalfor at least one sample entity wherein the signal is at least partiallyassociated with binding of the first and second binding partners (3525),and identifying the at least one cell of interest based at least in parton the measured signal (3526), to perform a one-bead assay.

The first plurality of particles may be provided suspended in aqueousmedia, and each particle of the first plurality of particles maycomprise a first binding partner that is specific to a second bindingpartner secreted by the at least one cell of interest. The first bindingpartner may, for example, be a polyclonal antibody (as described withreference to FIG. 32B). The second binding partner may be a bindingdomain of the antibody secreted by the cell of interest (as describedwith reference to FIG. 32B). The particles, cell sample, andencapsulation reagent may be provided in an emulsion, which may beincubated in order to analyze for a signal. A signal may then bemeasured (3525) based on a binding interaction that occurs between thefirst and second binding partners, which may result in a detectablecluster. The cluster may be visualized using a lensless imager such asthat described herein, or a microscope objective, for example. Thecluster may allow for the identification of the at least one cell ofinterest (3526), and may allow for the selection of a POD containing theat least one cell of interest. For example, one or more PODs containingthe at least one cell of interest may be sorted using the systems andmethods (e.g., electromerging, sorting) such as those described above.The assay may thus enable the selection of a POD containing the at leastone cell of interest for further processing of the cell of interest.

FIG. 35B depicts an exemplary method of selecting at least one cell ofinterest from a population of cells for use in a two-bead assay. Themethod may include providing an emulsion comprising a population ofcells, a first plurality of particles, and a second plurality ofparticles (3524 b), which may be an emulsion prepared according to themethod described when referencing FIG. 34B. Next, the method may includemeasuring a signal for at least one sample entity wherein the signal isat least partially associated with binding of the first and secondbinding partners and binding of the third and fourth binding partners(3525), and identifying the at least one cell of interest based at leastin part on the measured signal (3526), to perform a two-bead assay.

The first plurality of particles and the second plurality of particlesmay be provided suspended in aqueous media, and each particle of thefirst plurality of particles may comprise a first binding partner thatis specific to a second binding partner secreted by the at least onecell of interest. The first binding partner may, for example, be anantigen (as described with reference to FIG. 33C). The second bindingpartner may be a binding domain of the antibody secreted by the cell ofinterest (as described with reference to FIG. 33C). Each particle of thesecond plurality of particles may comprise a third binding partner thatis specific to a fourth binding partner secreted by the cell ofinterest. The third binding partner may, for example, be a monoclonalantibody (as described with reference to FIG. 33C). The fourth bindingpartner may, for example, be a binding domain of the antibody secretedby the cell of interest (as described with reference to FIG. 33C). Theparticles, cell sample, and encapsulation reagent may be provided in anemulsion, which may be incubated in order to analyze for a signal. Asignal may then be measured (3525) based on a binding interaction thatoccurs between the first and second binding partners, which may resultin a detectable cluster. The cluster may be visualized using a lenlessimager such as that described herein, or a microscope objective, forexample. The cluster may allow for the identification of the at leastone cell of interest (3526), and may allow for the selection of a PODcontaining the at least one cell of interest. For example, one or morePODs containing the at least one cell of interest may be sorted usingthe systems and methods (e.g., electromerging, sorting) such as thosedescribed above. The assay may thus enable the selection of a PODcontaining the at least one c ell of interest for further processing ofthe cell of interest.

In some variations, the population of cells and the first plurality ofparticles are encapsulated into a plurality of polydisperse sampleentities, and each particle of the first plurality of particles issuspended in aqueous media and comprises a first binding partner that isspecific to a second binding partner secreted by the at least one cellof interest.

In some variations, the method may further include providing a secondplurality of particles. The second plurality of particles may also beencapsulated into the polydisperse sample entities with the firstplurality of particles. Each particle of the second plurality ofparticles may comprise a third binding partner that is specific to afourth binding partner secreted by the at least one cell of interest(3527).

Exemplary Applications

FIG. 12 shows examples of various research and/or diagnosticapplications for the systems and methods described herein. In somevariations, the assay system may perform protein-based assays to detectvarious proteins. For example, the system may be used to detect markerssuch as Immunoglobulin G (IgG), alpha-fetoprotein (AFP), cancer antigens(e.g., CA125, CA 15-3), carbohydrate antigen (e.g., CA 19-9),carcinoembryonic gonadotropin (e.g., hCG or beta-hCG), prostate-specificantigen (PSA), and the like (e.g., for immunology-related research),Lactate Dehydrogenase (LDH) (e.g., to assess tissue damage such ascardiac stress and/or assess cancer, etc.), Beta 2 Microglobulin (B2M)(e.g., to help detect cancer), cytokines such as TNFα, IL-1, IL-2,IL-10, IL-12, type I interferons (e.g., IFN-α, IFN-β), IFN-γ,chemokines, and the like (e.g., to assess inflammation), andstreptavidin (e.g., to assess biotinylation etc), and the. The systemsdescribed herein may also perform cell-based detection assays. Forexample, the assay system may be used to detect white blood cells (e.g.,using anti-CD45 markers, such as for assessing leukemia and/or cancermetastasis), red blood cells (e.g., for hematology), and yeast cells(e.g., for characterizing expression vectors), etc. The assay system mayalso be used to perform expression-based assays. For example, the assaysystem may be used to detect expression of hybridomas, B-cells, andphage display, etc., such as for drug target identification.

Example 1

The system described herein was used to detect and differentiate betweenmicrospheres of multiple sizes. For example, FIG. 13A is an annotatedimage of micron bead particles (1310) encapsulated in PODS (1300)detected using computer vision. Samples containing 2 μm, 5 μm, 10 μm,and 15 μm glass beads were combined with an encapsulation reagent toencapsulate the sample into PODS. Each sample, containing PODS withmicron beads of a specific size, was introduced into the imaging chamberof the assay system. The lensless image sensors generated shadow imagesof the sample as it flowed through the chamber. The images were analyzedin order to detect the boundaries of the PODS (1300), and the micronbeads (1310). In addition to identifying the PODS and micron beads, thecomputer vision system measured the relative sizes of the micron beadswithin the PODS to generate a particle size score (PSS) (e.g., asdescribed below with respect to Examples 2-5). FIG. 13B is a graph ofthe distribution, the mean, and the median particle size scoresgenerated for each sample containing micron beads of a known size. FIG.13B shows, for example, the distribution of the particle size scores(1320) measured by the system in the sample containing 15 μm beads, anddenotes the mean (1330), and median (1340) of the particle score size.The distribution, the mean, and the median particle size scores measuredby the system are plotted for each of the 2 μm, 5 μm, 10 μm and 15 μmbead samples. Accordingly, the system may differentiate between 2 μm, 5μm, 10 μm, and 15 μm beads. The ability to differentiate within the 2-15μm size range may give the system the ability to perform various assays,such as those based on detection of protein and/or cell masses within aPOD.

Example 2

The system described herein were applied to perform quantitative proteinassays, for example, to quantify the concentration of IgG in a sample.For example, FIGS. 14A-14C are images showing computer vision detectionof IgG proteins (1400) in PODS (1410) at various concentrations. Toperform a quantitative protein assay of IgG, multiple samples, eachincluding a particular concentration of Rabbit IgG, were combined withantigen-conjugated to 1-2 micron latex beads specific to Rabbit IgG. Asshown in FIG. 15D, approximately 1 million PODS were generated from eachof seven samples, wherein each sample contained a different IgGconcentration ranging from 0 ng/mL to 480 ng/mL. Each IgG andantibody-conjugated bead mixture sample was vortexed with fluorocarbonoil to encapsulate the proteins into PODS. The binding of the antibodiesto the IgG proteins resulted in agglutination of IgG, and the formationof IgG protein masses (“agglutinates”) in the PODS. The PODS were thenintroduced into the imaging chamber of the assay system. As the PODSpassed through the chamber, the lensless image sensors generated shadowimages of the sample. Approximately 1 million PODS were analyzed in lessthan 10 minutes at each concentration. The images were analyzed todetect the protein masses within the PODS, and various parameters of thePODS and agglutinates were measured from the images, such as the sizeand shape of the PODS, and the grayscale values of the agglutinates. Theagglutinates present as darker on the shadow images, allowing detectionof agglutinates in the PODS.

For example, FIGS. 15A-15C depict POD parameters of POD area, PODradius, and POD circularity that were detected using computer visiontechniques for multiple tested concentrations of IgG. FIG. 15A shows thedistribution, the mean, and the median area of the PODS for each of thetested concentrations of IgG. FIG. 15B shows the distribution, mean, andmedian radius of the detected PODS in each tested concentration. FIG.15C shows the distribution, mean, and median of the circularity valuesof the detected PODS in each tested concentration.

Various POD parameter scores (“BE scores”) were derived from one or moremeasured parameters of the PODS and/or the features of interest in thePODS, such as sizes of aggregates, cells, particles, and/or changes inthose aggregates, cells, and/or particles within the PODS. For example,FIGS. 15E-15H illustrate examples of BE scores derived from a singularPOD parameter that can be detected from the images of the samplesgenerated by the lensless image sensors. FIG. 15E shows thedistribution, mean and median of BE Score 1, a measurement of the meangrayscale value of the detected aggregates within each POD at eachconcentration. FIG. 15F shows the distribution, mean and median of BEScore 2, a measurement of the grayscale standard deviation of thedetected aggregates within the PODS at each tested concentration. FIG.15G shows the distribution, mean, and median BE Score 3, a measurementof the grayscale minimum of the detected aggregates within the PODS ateach tested concentration. FIG. 15H shows the distribution, mean andmedian BE Score 4, a measurement of the grayscale maximum of thedetected aggregates within the PODS, at each tested concentration.

Furthermore, various POD parameter scores were derived as compositescores from multiple measured parameters of the PODS and/or the featuresof interest (e.g., aggregates, cells, other particles, etc.) within thePODS. Composite POD parameter scores may provide information (e.g.,trends in correlation with IgG concentration) that are not otherwiseavailable from POD parameter scores that are derived on a singlemeasured parameter. For example, FIGS. 16A-16D illustrate examples of BEscores calculated from a combination of multiple measured PODcharacteristics and/or BE scores that are derived from a singular PODparameter. A total of 11 BE scores were calculated from the images ofthe tested IgG samples described above, four of which were used tocorrelate image characteristics to IgG concentration, as depicted inFIGS. 16A-16D. BE Score 5 (shown in FIG. 16A) is generally a measure ofagglutination, based on a binary black and white image (e.g., objects inthe POD depicted as white, and background pixels are black, or viceversa). For example, BE Score 5 was based on a ratio of object pixels tobackground pixels, scaled to (e.g., divided by) POD area. BE Score 6(shown in FIG. 16B) is generally another measure of agglutination, basedon a distance transform of a binary black and white image such as thatdescribed above, where BE Score 6 was based on the pixel values of theresulting grayscale image, scaled to POD area. BE Score 7 (shown in FIG.16C) is generally a particle count score based on the number of detectedseparate objects in the POD. BE Score 8 (shown in FIG. 16D) is generallya particle size score, which relates to the average area of all theobjects identified in an imaged POD. Generally, BE Scores 5-8 were foundto increase with increased IgG concentration within a detection rangefor the experiment. As depicted in FIGS. 16A-16D, the detection rangefor IgG based on the BE scores was between about 30 and about 480 ng/mL.The graphs in FIGS. 16A-16D demonstrate that, within the IgG detectionrange, each of BE Scores 5-8 may be correlated with IgG concentration.Mathematical algorithms applied to BE Scores 5-8 were used to assess theconcentration of the IgG in each sample. Thus, because the size of theprotein masses within the PODS generally increases with proteinconcentration, the ability of the platform to measure the size of theprotein masses at the 2-15 μm level from the images of the PODS mayallow the platform to quantitate the protein concentration usingcharacteristics derived from images generated by lensless image sensors(e.g., based on empirical and/or calculated models correlating one ormore BE Scores to concentration, comparing one or more BE Scores to oneor more predetermined thresholds, etc.)

These results demonstrate that the parameters of PODS and agglutinatesmeasured from the shadow images of a sample may be used to quantitateprotein concentration in the samples based on a combination of one ormore BE scores such as those described above. Thus, the assay systemdescribed herein may be used to perform protein-based assays to quantifythe concentration of protein in a sample using antibody-conjugated beadsquickly and without the use of fluorescent labels.

Example 3

An inter-assay precision analysis was performed in a bead-mediated IgGassay using the system described herein. The inter-assay precisionanalysis enabled assessment of reproducibility and consistency of theassay systems described herein, as well as evaluation of sensitivity ofthe assay over different IgG concentrations. This information was usedto calculate margin of error at different protein concentrations. Forexample, an inter-assay precision analysis was performed by testingthree replicates (R1, R2, R3) of each IgG concentration used in theabove-described IgG assay. FIG. 17A depicts the ranges of BE Score 8, ameasurement of median particle size scores, in each of the threereplicates at each tested concentration. FIG. 17B depicts the ranges ofBE Score 2, a measurement of median grayscale standard deviation of thedetected aggregates within the PODS, in each of three replicates at eachtested concentration. As demonstrated by FIGS. 17A and 17B, differentlevels of the IgG concentration exhibited different levels of precisionby which these two parameters may be measured. For example, FIGS. 17Aand 17B demonstrate that for at least some BE Scores, sensitivity of theassay system varies with concentration of the detected analyte, atdifferent segments of the detected range. For example, FIGS. 17A and 17Bsuggest that the precision of the particle size score median andgrayscale standard division median measurements generally decreases asIgG concentration increases (the range of the values increases withincreased concentration).

Example 4

The system described herein was used to test a rabbit IgG assay todetermine if bovine serum interfered with the specificity of the assay.A control sample and an experimental sample were separately analyzedwith the assay system. The control sample included 500-fold dilutedbovine serum. The control sample was then introduced into the imagingchamber of the system, and the lensless image sensors generated shadowimages of PODS including the control sample (FIG. 18A). The experimentalsample was prepared by mixing 960 ng/mL rabbit IgG serum with 500-folddiluted bovine serum and antibody-conjugated beads specific to IgG, andcombined with encapsulation reagent (e.g., surfactant) to encapsulatethe IgG proteins into PODS. The experimental rabbit IgG sample wasintroduced into the imaging chamber of the system, and the lenslessimage sensors generated shadow images of PODS including the experimentalsample (FIG. 18B).

FIG. 18C depicts the distribution, mean, and median of BE Score 8(particle size score, as described above with respect to FIG. 16D), forthe control and experimental samples. The difference in thedistribution, mean and median of BE Score 8 between the control sampleand the experimental sample demonstrated that rabbit IgG does not showsignificant cross-species reactivity with bovine serum. In other words,this example suggests that bovine serum may not interfere with thespecificity of the assay.

Example 5

The system described herein was successfully used in cell-baseddetection assays (e.g., cell type and cell count assays). For example,the assay system described herein was used to detect the presence ofleukocytes in a sample, as illustrative of other cell-based detectionassays. As shown in the schematic illustration of FIG. 19A, a CD-45+leukocyte (white blood cell) was tagged or “decorated” with anti-CD45nanoparticles (e.g., glass beads). To identify the presence of CD-45+leukocytes in a sample, the sample containing CD-45 cells was mixed withanti-CD45 nanoparticles. The nanoparticles selectively bound to theunique surface markers on the CD-45 cells, which provided the means todifferentiate the CD-45 cells from other cells. The sample including theCD-45 cells bound to nanoparticles was mixed with an encapsulationreagent and vortexed to encapsulate the CD-45 cells into PODS. Thebinding of the anti-CD45 nanoparticles to the CD-45 cells resulted inagglutination of the CD-45 cells, and the formation of CD-45agglutinates in the PODS. The PODS were then introduced into the imagingchamber of the device, and the lensless image sensors generated shadowimages of the PODS as they passed through the chamber. The computervision system analyzed the images of the PODS (FIG. 19B) to detect thepresence of the CD-45 cells based on the increased greyscale values ofthe CD-45 agglutinates. The CD-45 cell agglutinates (1910) in the PODS(1900) present as darker in the shadow image compared to other cells,which allows for detection and enumeration of the CD-45 cells by thecomputer vision system. The use of the assay system to identifynanoparticle-tagged CD-45 cells demonstrates that the system may performcell detection and enumeration based on selective binding ofnanoparticles to cell surface markers. It may be inferred that thesystem may also be used to perform detection and enumeration of variouscells that exhibit surface markers.

Example 6

The system described herein can also be used to quickly and efficientlydistinguish between dead and live cells in a sample. For example, theassay system described herein was used to detect and/or enumerate deadyeast cells. FIG. 20A depicts computer vision detection of PODS (2000)containing dead yeast cells (2010). To perform a dead-cell count assay,yeast cells were stained with trypan blue, and encapsulated with areagent into PODS. The sample of PODS containing the stained yeast cellswas introduced into the imaging chamber of the assay system, and theimaging system generated shadow images of the PODS as they passedthrough the chamber. The computer vision system identified the deadyeast cells in the shadow images, based on the fact that the dead yeastcells absorbed more of the blue dye due to their increased porositycompared to live cells. The stained yeast cells present as darker in theshadow image, allowing the computer vision system to identify thecolor-saturated dead yeast cells based on a decreased grayscale value.The computer vision system analyzed the images to provide a dead cellparticle count score as shown in FIG. 20B, and a particle size score, asshown in FIG. 20C. The ability of the assay system to provide a deadcell count and size score of dead yeast cells demonstrates that thesystem may detect, enumerate, and/or measure dead cells based on the useof cell staining techniques. This ability may be extrapolated to performenumeration of various cells types using differentiation of cells basedon staining. Furthermore, after distinguishing between live and deadcells, live and dead cells may be sorted using fluidic systems asdescribed herein (e.g., to dispense cells of interest into a suitablevessel such as a well plate for further analysis and/or culturing).

Example 7

Additionally or alternatively, a method for processing a sample mayinclude detecting one or more cell secretions in a sample (or the cellsthemselves). For example, generally, in a cell secretion assay, one ormultiple analytes (e.g., a protein of interest such as a cytokine or amonoclonal antibody (mAb)) may be secreted by one or more cells, and itmay be desirable to determine which analyte(s) are secreted. Withreference to FIG. 22, a sample including secreting cells and at leastone detection reagent may be dispersed into PODS and passed through anassay system as described above to produce shadow images of the PODS.The one or more analytes of interest that are secreted from the cellsmay be specific to a detection reagent, such that the resultingaggregation results in a darkened, shadowed mass that is detectable inthe shadow image. Thus, identification of an aggregated mass in a PODmay indicate that one or more analytes of interest has been secretedfrom the cells. Multiple analytes (e.g., specific to different reagentsmixed with the sample) may furthermore be identifiable in parallel usingthe assay system.

Example 8

Hybridoma cells may be produced by injecting a specific antigen into amouse, collecting antibody-producing B-cells from the mouse, and fusingthe B-cells with tumor cells to make them “immortal.” It is valuable toidentify and collect hybridoma cells that produce a significant amountof the desired antibody. For example, hybridoma cells that produce asignificant amount of IgG antibodies (Ab), where the IgG Ab are specificand have high affinity and/or specificity to certain antigens, arevaluable cells to identify and collect for therapeutic purposes.However, conventional protocols for screening such high secretorhybridoma cells are costly and time-consuming, as the hybridoma cellsmust be replicated and multiplied over a number of days under acarefully controlled environment.

In one example, a method for processing a sample using a chamber andimager array system, such as that described herein, may be used toidentify hybridoma cells that are high secretors or producers ofantibodies for specific targets. A sample was prepared by vortexinghybridoma cells (mouse cells) with anti-mouse IgG pAb coupled 1 μmpolystyrene beads, along with a carrier oil with surfactant, to form anemulsion including PODS, where each of the PODS were loaded with morethan one cell (such that average number of cells per POD λ>1). Thesample was imaged using a chamber and imager array system describedabove at t=0, t=1 hour (after 1 hour incubation), and t=4 hours (after 4hours incubation). As shown in these images (FIG. 27, at both 4×objective and 10× objective magnification), large clusters in the PODSwere observable after only 1 hour, and further observable after 4 hours.Thus, the images of FIG. 27 suggest that hybridoma cells secrete IgG ina detectable range within PODS even after a brief incubation period ofonly 1 hour, such that hybridoma cells with a sufficiently strongbiosignal (e.g., manifested as identifiable clumping in images) may beidentified in PODS. In some variations, such high secretor hybridomacells may further be sorted and collected as an output with a highconcentration of high secretor hybridoma cells.

Example 9

FIG. 29A is a table of exemplary system parameters for an exemplaryvariation of an electromerging chamber arrangement and exemplary samplewith PODS. The exemplary sample included a mixture of (i) the indicatedvolume of aqueous cell media with approximately 10 million B cells and(ii) the indicated volume of carrier oil with surfactant, leading to thecomposition outlined in FIG. 29A, including approximately 154 milliontotal PODS having the indicated POD characteristics. Some of the PODS inthe sample were smaller than the 35 μm chamber gap spacing, and were bespherical (not flattened into an oblate shape) when in the chamber. Dueto the shape of these PODS, it would be more difficult to detect thecells contained in them, and may cause cells of interest to beinadvertently lost or discarded.

Probabilistic modeling was performed to estimate the number of cellsthat are not detected by an electromerging chamber arrangement. Thedistribution of actual POD diameters for the sample is shown in FIG.29B, and may be modeled closely as a Gamma distribution (FIG. 29C).Using probabilistic modeling in view of the average POD characteristicsindicated in FIG. 29A, the expected percentage of “small” PODS (that is,smaller than 35 μm and having a volume of less than 22 pL) that would beoverlooked, but are not actually empty, is about 0.017%. In other words,assuming 1 cell maximum per overlooked POD, processing the sample in thechamber would result in potentially failing to detect approximately 170cells per million PODS, or about 2615 cells total. Given a total B cellpopulation in the original sample of 15.4 million, this meanspotentially failing to detect about 0.017% of the cells in the sample.Thus, the analysis with respect to the sample and system associated withFIGS. 29A-29C suggests that only a negligible proportion of cells may beinadvertently undetected using the chamber described herein.

Example 10

As described above, a one-bead and/or two-bead clustering assay asdescribed herein may be performed to identify a particular cell ofinterest within a population of cells. The cell of interest may be ahigh secretor of a target analyte, such as an antibody, or may be a highsecretor of a target analyte having a high affinity for an antigen.Experimental tests were performed to demonstrate that B cells and CHOcells can generate a measurable and detectable signal in PODS within aspecified time frame using the one-bead or two-bead assays describedherein.

FIGS. 36A-36C depict 4× objective microscope cell images from tests of aone-bead assay. To demonstrate the one-bead assay, one batch ofanti-mouse IgG polyclonal (pAb) beads (FIG. 36A) and two batches ofanti-human IgG pAb beads (FIGS. 36B-36C) were prepared for performing aone-bead assay. FIGS. 36A-36C show that all batches of beads showedclustering when 10 μg/ml of mouse or human IgG were present. Each batchis shown against a no cell (NC) control.

FIG. 37 depicts images from tests performed using a one-bead assay asdescribed herein, used to assess mouse IgG secreting single hybridomacells in PODS. Microscope images at both 4× and 10× objectives are shownat time=0, t=1 hour, t=2 hours, t=4 hours, and t=6 hours afterincubation. The images show clustering at all time points beginning fromt=1 hour.

FIG. 38 depicts images from tests performed using a two-bead assay asdescribed herein, used to assess antigen-specific antibody secretingsingle hybridoma cells in PODS. Bovine IgM antigen specific antibodieswere analyzed in the tests. Microscope images at both 4× and 10×objectives are shown at time=0, t=1 hour, t=3 hours, and t=5 hours afterincubation. The images show clustering at all time points beginning fromt=1 hour.

Tests were performed to check for non-specific background clusteringusing a cell line that secretes antibodies that are not against thespecific antigen. Hybridoma cells that secrete anti-bovine insulin(HB-123 cell line) were used as a control in a bovine IgM antigentwo-bead assay. Bovine IgM antigen beads were used. The CRL-1894 cellline was also used to test for specific clustering, with the CRL-1894cells being known to secrete the anti-bovine IgM monoclonal antibody).0.005% trypan blue was added to monitor cell viability throughout thetests.

FIG. 39A depicts 4× objective microscope images from the tests conductedusing the HB-123 cell line, wherein no clustering was expected. Theimages show that at time=0, t=1 hour, and t=3 hours, no backgroundclustering occurred. In contrast, FIGS. 39B-39D show 10× objectivemicroscope images at time=0, t=1 hour, and t=3 hours, showing thatclustering occurred starting from t=1 hour. The cell viability wasmeasured to be about 84%.

The foregoing description, for purposes of explanation, used specificnomenclature to provide a thorough understanding of the invention.However, it will be apparent to one skilled in the art that specificdetails are not required in order to practice the invention. Thus, theforegoing descriptions of specific embodiments of the invention arepresented for purposes of illustration and description. They are notintended to be exhaustive or to limit the invention to the precise formsdisclosed; obviously, many modifications and variations are possible inview of the above teachings. The embodiments were chosen and describedin order to explain the principles of the invention and its practicalapplications, they thereby enable others skilled in the art to utilizethe invention and various embodiments with various modifications as aresuited to the particular use contemplated. It is intended that thefollowing claims and their equivalents define the scope of theinvention.

The invention claimed is:
 1. A system for processing a sample comprisinga plurality of sample entities, the system comprising: a chamberconfigured to accommodate the sample, wherein the chamber comprises: atleast one electrode configured to deliver electrical energy sufficientto merge a selected portion of sample entities in the sample; and asorting arrangement configured to separate sample entities of the samplebased on size of the sample entities, wherein the sorting comprises aplurality of spacers arranged in a staggered array.
 2. The system ofclaim 1, wherein the chamber is configured to accommodate atwo-dimensional flow of the sample.
 3. The system of claim 1, whereinthe chamber comprises a plurality of electrodes extending between firstand second opposing surfaces of the chamber.
 4. The system of claim 1,wherein the sorting arrangement is a passive sorting arrangement.
 5. Thesystem of claim 4, wherein the chamber comprises a first outlet and asecond outlet, wherein the first outlet is sized to pass substantiallyonly sample entities below a predetermined threshold sample entity size,and the second outlet is sized to pass sample entities above thepredetermined threshold sample entity size.
 6. The system of claim 1,wherein the sample comprises a plurality of PODS.
 7. The system of claim1, further comprising an imager array comprising at least one lenslessimage sensor, the imager array being configured to generate one or moreimages of the sample in the chamber, and a controller configured toactivate the at least one electrode to deliver electrical energy to theselected portion of sample entities based on the one or more images ofthe sample.
 8. A system for processing a sample, the system comprising:a chamber configured to accommodate the flow of a sample, wherein thechamber comprises at least one electrode configured to selectivelydeliver electrical energy to at least a portion of the sample; an imagerarray configured to image the flow of the sample in the chamber; and acontroller configured to activate the at least one electrode based on ananalysis of one or more images from the imager array.
 9. A method forprocessing a sample comprising a plurality of sample entities, themethod comprising: receiving a sample in a chamber comprising at leastone electrode and a sorting arrangement comprising a plurality ofspacers arranged in a staggered array; characterizing one or more sampleentities in the sample as discard sample entities; merging the discardsample entities by delivering electrical energy from the at least oneelectrode to the discard sample entities; and sorting sample entities ofthe sample based on size of the sample entities.
 10. The method of claim9, wherein characterizing one or more sample entities comprisesreceiving one or more images of the sample in the chamber andcharacterizing one or more sample entities based on the one or moreimages, wherein the one or more images comprises an optical image. 11.The method of claim 10, wherein the optical image is a shadow image ofthe sample.
 12. The method of claim 9, wherein delivering electricalenergy comprises activating a pair of electrodes in accordance with awaveform.
 13. The method of claim 9, wherein sorting sample entitiescomprises permitting sample entities of a first size to pass through afirst outlet of the chamber, and permitting sample entities of a secondsize to pass through a second outlet of the chamber.
 14. The method ofclaim 9, wherein the sample entities in the sample are PODS.
 15. Themethod of claim 9, wherein at least a portion of the sample entitiescontains one or more cells secreting a substance of interest, andcharacterizing one or more sample entities in the sample comprisescharacterizing secretion levels of the one or more cells.
 16. The methodof claim 15, wherein characterizing secretion levels comprisescharacterizing agglutination in the one or more cells.
 17. The system ofclaim 8, wherein the chamber is configured to accommodate atwo-dimensional flow of the sample.
 18. The system of claim 8, whereinthe chamber comprises a plurality of electrodes extending between firstand second opposing surfaces of the chamber.
 19. The system of claim 8,further comprising a sorting arrangement configured to separate sampleentities in the sample based on size of the sample entities.
 20. Thesystem of claim 8, wherein the chamber comprises a first outlet and asecond outlet, wherein the first outlet is sized to pass substantiallyonly sample entities in the sample below a predetermined thresholdsample entity size, and the second outlet is sized to pass sampleentities in the sample above the predetermined threshold sample entitysize.